Identification of pathogen bacteria and protozoa in treated urban wastewaters discharged in the Ebro River (Spain): water reuse possibilities.

The aim of this research work is to identify the presence of pathogens, bacteria and protozoa, in different treated urban wastewaters and to relate biological pollution with the processes used in wastewater treatment plants. A study of the possibilities for water reuse is carried out taking into account bacterial and parasite composition. The analysed bacteria and protozoa are: Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium perfringens (spore), Salmonella spp., Legionella spp., helminths eggs, Giardia, Cryptosporidium spp. and free-living amoebae (FLA). The selected municipal wastewater treatment plants (MWTPs) are located in Navarra (Spain) and the main difference between them is the use of natural lagoons as tertiary treatment in some plants. The results concerning bacteriological identification showed contamination of mainly faecal origin, and the use of natural lagoons as tertiary treatment in some MWTPs produced an important disinfection effect. Moreover, pathogen parasites such as Giardia and Cryptosporidium were not detected in the samples studied although FLA were identified in all cases.

Evaluation of an immunochromatographic dip strip test for simultaneous detection of Cryptosporidium spp, Giardia duodenalis, and Entamoeba histolytica antigens in human faecal samples.

Immunochromatographic (IC) tests may play an important role in the future diagnosis of parasitic diseases because of their speed and simplicity of use. A recently developed test to detect Cryptosporidium spp, Giardia duodenalis and Entamoeba histolytica was evaluated. Microscopy and PCR were the "gold standard" reference techniques and the results of this IC test were compared with those obtained with ELISA and IC single test for the three parasites. One hundred sixty stool samples were assayed. Using microscopy, 22 samples were diagnosed as positive for Cryptosporidium spp., 31 for Giardia duodenalis, 41 for Entamoeba histolytica/dispar, and 68 had a negative diagnosis for the three parasites. Results of IC tests show sensitivities of 70-72% for Cryptosporidium, 90-97% for Giardia and 62.5% for Entamoeba histolytica. Specificities were of 93.6-94.9%, >99% and 96.1%, respectively. In all diagnoses, agreement with microscopy and PCR was over 90%, except in the triple test and microscopy in E. histolytica detection that was 76.3%, due to the inability of microscopy to differentiate E. histolytica from nonpathogenic species such as E. dispar or E. moshkovskii. The triple stool immunoassays provide adequate sensitivities and specificities for use in outbreak situations, for screening proposals and for massive assays in endemic areas where a large number of samples must be analysed or as complementary test for individual diagnosis.

The needs of families who care for individuals with kidney failure on comprehensive conservative care: A qualitative systematic review.

BACKGROUND: Integrating the family of patients with kidney failure on comprehensive conservative care could benefit patients, families, and the health care system. However, there is a knowledge gap in this phenomenon since no systematic review has focused on the families' needs who care for individuals with kidney failure on comprehensive conservative care. OBJECTIVES: To understand the primary needs of families who care for people with kidney failure on comprehensive conservative care. METHOD: A systematic literature review of qualitative studies, followed by a content analysis was carried out. PubMed, CINAHL, and PsycINFO databases were used to search for articles published in English and Spanish between 2010 and 2021. The ENTREQ guideline was used for reporting. RESULTS: Five relevant studies were included in this study. The analysis has allowed identifying key aspects of knowledge, psychological, social and spiritual needs of family members of patients with kidney failure on comprehensive conservative care. CONCLUSIONS: This systematic review has revealed that families experience a lack of information and continuity of care by health care professionals. Added to this is the psychological burden they bear due to the feeling of indefinite care in time and uncertainty about the death of their loved one. All this, without the necessary support from their immediate family environment and social institutions. In light of these data, a paradigm shift in society and the health care received by these families is essential.

Assessment of the methodologies used in microbiological control of sewage sludge.

Sewage sludge usually contains potentially polluting substances such as heavy metals, organic pollutants and various organisms including bacteria, protozoa, helminths, viruses and algae, some of which may be pathogenic. Certain of these pathogens could be transferred to the soil if the sludge is used on agricultural or land recovery applications. For its application on agricultural land, sewage sludge must comply with the limits established in the legislation, which in Europe does not include quality standards regarding microbiological parameters. Nevertheless, the presence of pathogens could limit its agricultural use, as it could pose a risk to human, animal and environmental health. This study compares 4 different methodologies used in microbiological analysis in order to identify the most efficient and reliable method on determining bacteria in sewage sludge. Escherichia coli and Enterococcus faecium are used as bacterial indicators. The results obtained in this work indicate that results obtained with three different plate count methods cannot be comparable with those obtained with the MPN method. The membrane filtration method is recommended for its high precision and sensitivity, both in low and high bacterial loads. It is also concluded that it would be necessary to establish the quality standard in concordance with the method used.

In-vitro development of an effective treatment for Acanthamoeba keratitis.

The aim of this study was to develop an in-vitro topical treatment for Acanthamoeba keratitis (AK) effective against cysts and trophozoites. Qualitative assays were performed with voriconazole, chlorhexidine, propamidine, cellulase, tobramycin, ciprofloxacin and paromomycin as monotherapy and various combinations. Riboflavin with ultraviolet-A (R + UV-A) as monotherapy or combined with voriconazole and moxifloxacin was also tested. Quantitative assays to assess cyst viability after treatment were performed for the chemicals that showed the highest activity in the qualitative assays. Paromomycin and propamidine did not show antiamoebic activity. Regardless of the total dose, no amoebicidal effect was observed for R + UV-A. Tobramycin, ciprofloxacin, voriconazole, chlorhexidine and cellulase were selected for quantitative assays because they appeared to cause greater damage to the structure of amoebae. Chlorhexidine and ciprofloxacin were the most active against Acanthamoeba spp. as monotherapy. Among the combinations evaluated, ciprofloxacin-voriconazole-chlorhexidine showed the greatest amoebicidal activity, with severe damage of the cellular membrane and an important decrease in cell concentration. In summary, ciprofloxacin as monotherapy and in combination with voriconazole and chlorhexidine has been classified as promising treatment. Additional in-vivo studies in animal models and clinical trials in patients with AK should be considered to confirm the efficacy of ciprofloxacin.

[A new case of psychotic break as the presenting symptom of anti-NMDA receptor encephalitis. Treatment from a neuropsychiatric approach]. / Nuevo caso de brote psicotico como presentacion de la encefalitis antirreceptor de NMDA. Enfoque de tratamiento desde la neuropsiquiatria.

INTRODUCTION: Limbic encephalitis generated by anti-N-methyl D-aspartate (NMDA) receptor antibodies is a severe acute neurological condition that is more prevalent in young females and is associated with an underlying tumour. The appearance of severe initial psychiatric signs and symptoms and the fact that it is accompanied by emotional disorders and severe cognitive impairment make it necessary to reconsider the contributions of neuropsychiatry to the diagnosis, development and treatment of the disease, since these have received little attention from researchers to date. CASE REPORT: We report on what is, to our knowledge, the only case in Navarra, associated with an ovarian teratoma, with special attention paid to its assessment, intervention and neuropsychiatric development. CONCLUSION: Anti-NMDA receptor encephalitis is a disease that is usually accompanied by predominant psychiatric manifestations at the outset of the illness, together with cognitive deterioration that commonly affects memory and the executive functions. Treatment consisting in beginning a process of rehabilitation that includes compensatory strategies for deficits, cognitive stimulation and psychopharmacological treatment in the recovery phase has proved to be effective.

TITLE: Nuevo caso de brote psicotico como presentacion de la encefalitis antirreceptor de NMDA. Enfoque de tratamiento desde la neuropsiquiatria.Introduccion. La encefalitis limbica generada por anticuerpos antirreceptor de N-metil-D-aspartato (NMDA) es una entidad neurologica aguda y grave, mas prevalente en mujeres jovenes y asociada a un tumor subyacente. La aparicion de sintomatologia psiquiatrica inicial grave y el curso con alteraciones emocionales y deficit cognitivo grave hacen necesario reflexionar sobre las aportaciones, poco estudiadas hasta el momento, de la neuropsiquiatria al diagnostico, la evolucion y el tratamiento de la enfermedad. Caso clinico. Se presenta el unico caso del que se tiene constancia en Navarra, asociado a un teratoma ovarico, atendiendo especialmente a la evaluacion, la intervencion y la evolucion neuropsiquiatrica. Conclusion. La encefalitis antirreceptor de NMDA es una enfermedad que suele cursar con manifestaciones psiquiatricas predominantes en el inicio de la enfermedad, asi como con deterioro cognitivo que afecta habitualmente a la memoria y a las funciones ejecutivas. Se demuestra eficaz iniciar un proceso de rehabilitacion que incluya estrategias compensatorias del deficit, estimulacion cognitiva y tratamiento psicofarmacologico en la fase de recuperacion.

[Proposed model of executive functions based on factorial analyses]. / Propuesta de un modelo de funciones ejecutivas basado en analisis factoriales.

INTRODUCTION: Since Lezak coined the term executive functions to refer to the mental capabilities that are considered essential for performing efficacious, creative and socially acceptable behaviour, they have gradually grown in importance in neuropsychological research. Different models have been proposed to explain their nature, but there is no general agreement as to whether we are dealing with a unitary construct or a multimodal processing system with independent, but interconnected, components. With the aim of gaining a deeper knowledge of the structure of this construct, researchers have conducted lesion, neuroimaging and, more recently, factorial analysis studies, the latter being seen as a promising methodology for expanding our knowledge about such a generic concept as the executive functions. DEVELOPMENT: The purpose of this study is to carry out a systematic review of factorial models of attention and executive control in adults, between the years 1991 and 2016, using the PubMed, OvidSP and PsycINFO databases. Altogether, 33 papers were reviewed. Based on the literature, an integrating proposal of the executive functions is put forward. CONCLUSIONS: Although we do not have just one single model that can account for the complexity of the executive functions, there does seem to be general agreement on their multidimensionality. In factorial analyses, there is strong evidence of updating, inhibition and alternation, although there are also studies that propose novel factors. Our integrating proposal aims to combine the executive processes found in the literature with their corresponding neuroanatomical correlates, and defends the stance that the ideal methodology should use information from lesion studies, neuroimaging techniques and psychometric-computational models.

TITLE: Propuesta de un modelo de funciones ejecutivas basado en analisis factoriales.Introduccion. Desde que Lezak acuñara el termino de funciones ejecutivas como las capacidades mentales esenciales para llevar a cabo una conducta eficaz, creativa y aceptada socialmente, estas han adquirido progresivo protagonismo en la investigacion neuropsicologica. Diversos modelos han sido planteados para explicar su naturaleza, pero no existe consenso respecto a si nos encontramos ante un constructo unitario o un sistema de procesamiento multimodal con componentes independientes, pero interconectados. Para conocer la estructura de este constructo se han utilizado estudios de lesion, neuroimagen y, recientemente, el analisis factorial, que se plantea como una metodologia prometedora para ampliar nuestro conocimiento sobre un concepto tan generico como las funciones ejecutivas. Desarrollo. El proposito de este estudio es realizar una revision sistematica de modelos factoriales de atencion y control ejecutivo en adultos, entre los años 1991-2016, utilizando las bases de datos PubMed, OvidSP y PsycINFO. En total, se revisaron 33 articulos. A partir de la bibliografia, se realiza una propuesta integradora de los procesos ejecutivos. Conclusiones. Aunque no disponemos de un unico modelo que pueda explicar la complejidad de las funciones ejecutivas, si parece existir acuerdo respecto a su multidimensionalidad. En analisis factoriales, actualizacion, inhibicion y alternancia gozan de fuerte evidencia, si bien hay trabajos que plantean factores novedosos. Nuestra propuesta integradora trata de combinar los procesos ejecutivos hallados en la bibliografia con sus correspondientes correlatos neuroanatomicos, defendiendo que la metodologia ideal deberia utilizar informacion procedente de estudios de lesion, tecnicas de neuroimagen y modelos psicometricos-computacionales.

Genetic relatedness and antimicrobial resistance determinants among clinical isolates of enterococci from Cuba.

This study describes the genetic relationships and antimicrobial resistance determinants found among 99 clinical isolates of enterococci from 15 different hospitals in Cuba. Pulsed-field gel electrophoresis SmaI analysis demonstrated a high degree of genetic diversity. A limited number of multiresistant Enterococcus faecalis clones, showing resistance to three or more families of antimicrobial agents, were detected simultaneously in different institutions, suggesting inter-hospital circulation of selected clones, and/or selection of particular clones following their introduction into the hospital environment. Antimicrobial resistance determinants, including erm(B), aac(6')-aph(2'), aph(3'), ant(6), vanB (E. faecalis) and vanA (Enterococcus faecium) were detected by PCR in various isolates.

Bioaccumulation of pathogenic bacteria and amoeba by zebra mussels and their presence in watercourses.

Dreissena polymorpha (the zebra mussel) has been invading freshwater bodies in Europe since the beginning of the nineteenth century. Filter-feeding organisms can accumulate and concentrate both chemical and biological contaminants in their tissues. Therefore, zebra mussels are recognized as indicators of freshwater quality. In this work, the capacity of the zebra mussel to accumulate human pathogenic bacteria and protozoa has been evaluated and the sanitary risk associated with their presence in surface water has also been assessed. The results show a good correlation between the pathogenic bacteria concentration in zebra mussels and in watercourses. Zebra mussels could therefore be used as an indicator of biological contamination. The bacteria (Escherichia coli, Enterococcus spp., Pseudomonas spp., and Salmonella spp.) and parasites (Cryptosporidium oocysts and free-living amoebae) detected in these mussels reflect a potential sanitary risk in water.

[Contributions of neuropsychology to anti-NMDA receptor antibody encephalitis: a literature review]. / Aportaciones de la neuropsicologia a la encefalitis por anticuerpos antirreceptor de NMDA: revision de la bibliografia.

Limbic encephalitis generated by anti-N-methyl-D-aspartate (NMDA) receptor antibodies is an acute and severe neurological entity, which is more prevalent in young females and is associated to an underlying tumour. Since it leads to severe cognitive impairment, thought needs to be given to the contributions of neuropsychology to the diagnosis, development and treatment of the disease, which have received little attention from researchers to date. A review is conducted of the prior literature, evaluating the measurement of the cognitive symptoms (predominantly mnemonic and executive) associated to this disease. Valid, reliable neuropsychological instruments are proposed, and it is suggested that neuropsychological measures may be used as parameters to follow up these patients which help monitor their functionality in daily living once they have recovered from the acute phase. Similarly they can become a basis on which to assemble rehabilitation programmes that favour the accomplishment of personal autonomy and the patients' reintegration in the community. Nevertheless, we stress the need to include neuropsychologists and neuropsychiatrists in not only the detection but also the treatment of these patients so as to enable them to recover their personal independence and re-adapt to their natural settings.

TITLE: Aportaciones de la neuropsicologia a la encefalitis por anticuerpos antirreceptor de NMDA: revision de la bibliografia.La encefalitis limbica generada por anticuerpos antirreceptor de N-metil-D-aspartato (NMDA) es una entidad neurologica aguda y grave, mas prevalente en las mujeres jovenes y asociada a un tumor subyacente. El curso con deficit cognitivo grave hace necesario reflexionar sobre las aportaciones de la neuropsicologia al diagnostico, evolucion y tratamiento de la enfermedad, poco estudiadas hasta el momento. Se revisa la bibliografia precedente, criticando la medicion de los sintomas cognitivos (predominantemente mnesicos y ejecutivos) asociados a esta enfermedad. Se proponen instrumentos neuropsicologicos validos y fiables, y se plantea que las medidas neuropsicologicas pueden servir como parametros de seguimiento de estos pacientes que ayuden a monitorizar su funcionalidad en la vida diaria una vez recuperados de la fase aguda, asi como convertirse en una base sobre la que articular programas de rehabilitacion que favorezcan el logro de la autonomia personal y la reinsercion comunitaria de los pacientes. Con todo, se subraya la necesidad de incluir a neuropsicologos y neuropsiquiatras no solo en la deteccion, sino en el tratamiento de los pacientes, en pro de alcanzar la recuperacion de la independencia personal y la readaptacion a sus entornos naturales.

Analysis of the mechanism of quinolone resistance in nalidixic acid-resistant clinical isolates of Salmonella serotype Typhimurium.

Over a period of 2.5 years, 42 cases of gastro-enteritis caused by nalidixic acid-resistant Salmonella serotype Typhimurium occurred in Malaga. The epidemiological relationship among the strains involved was investigated by analysis of plasmid profile and of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). Despite having different plasmid profiles, all 42 nalidixic-acid resistant Typhimurium isolates had evolved from one clone as shown by analysis of chromosomal DNA by PFGE. The mechanism of quinolone resistance in these Typhimurium isolates was also investigated. Analysis of outer-membrane proteins and lipopolysaccharide from quinolone-susceptible and resistant clinical isolates tested showed no differences. All nalidixic acid-resistant isolates had MICs for ciprofloxacin of 0.25 mg/L and for nalidixic acid of 1024 mg/L. Polymerase chain reaction fragments of 285 bp, containing the quinolone resistance-determining region of the gyrA gene, and of 237 bp, containing the region of parC homologous to the quinolone resistance-determining region of the gyrA gene, were sequenced. All resistant isolates presented a change at Ser-83 to Phe in the GyrA protein, but no changes were observed in the ParC protein. These findings indicated that this mutation in gyrA plays a major role in the acquisition of nalidixic-acid resistance in clinical isolates of Typhimurium.

Antibiotic resistance and epidemiological typing of Staphylococcus aureus strains from ovine and rabbit mastitis.

Mastitis is a serious problem for sheep and rabbit farms, Staphylococcus aureus being the main causal agent. Fifty strains of S. aureus isolated from sheep and rabbits from farms located in diverse geographical regions of Spain were studied. Their resistance pattern and plasmid profile was related to the pulsotypes obtained by pulsed-field gel electrophoresis (PFGE). The results showed great heterogeneity in staphylococci isolated from sheep, both in pulse-type and plasmid profile. We found in addition, antibiotic-resistant strains and aminoglycoside-modifying enzyme (AGMEs) producer strains. The genotypes corresponding to staphylococci isolated from rabbits were less heterogeneous, although they also could be subdivided by plasmid profile and resistance patterns. Resistance to antibiotics such as methicillin or AGMEs production could indicate possible human origin of the strains or a possible source of resistant strains for human beings.

Macrolide resistance phenotypes of commensal viridans group streptococci and Gemella spp. and PCR detection of resistance genes.

One hundred and sixty viridans group streptococci (VGS) and 26 Gemella spp. resistant to erythromycin were studied to detect macrolide lincosamide and streptogramin B (MLS(B)) phenotypes and to investigate resistance rates to other antibiotics. The M phenotype was most prevalent in both bacterial groups (59.6% in VGS, 69.2% in gemellae) and the iMLS(B) phenotype was found least often (9.3 and 13.9%, respectively). All isolates with M phenotype had the mef(A/E) gene, being prevalent the mef(E) subclass. cMLS(B) and iMLS(B) strains contained the erm(B) gene, alone or in combination with the mef(A/E) gene. Thirteen isolates were intermediately resistant to quinupristin/dalfopristin and 11 strains showed low susceptibility to telithromycin. Linezolid was active against all the isolates tested and tetracycline resistance was the major one in VGS (41.6%) and Gemella spp. (46.2%).

In vitro activity of telithromycin, quinupristin/dalfopristin, linezolid and comparator antimicrobial agents against Staphylococcus aureus clinical isolates.

We have studied the prevalence of the different macrolide, lincosamide, streptograminB (MLS(B)) phenotypes among clinical Staphylococcus aureus isolates erythromycin- and/or oxacillin-resistant; and also the activity of other antimicrobial agents including telithromycin, quinupristin/dalfopristin, linezolid, aminoglycosides, chloramphenicol and vancomycin. We found that 64.86% of S. aureus were oxacillin-resistant. While the most prevalent MLS(B) phenotype among methicillin-resistant S. aureus (MRSA) was constitutive MLS(B) (cMLS) (83%), among methicillin-susceptible S. aureus (MSSA) it was inducible MLS(B) (iMLS(B)) (90%). Kanamycin resistance was more frequent than resistance to other aminoglycosides, being 100% for MRSA. Telithromycin was only active against iMLS(B), MS and erythromycin-susceptible isolates, although resistance rates were found among iMLS(B) MSSA (2.78%). Quinupristin/dalfopristin showed greater activity, with resistance rates of 2.5% for MRSA and 1.53% for MSSA. Both vancomycin and linezolid were fully active against all the isolates tested, with the highest MIC value being 2 microg/ml and 4 microg/ml, respectively. Among MRSA strains, 81.67% displayed resistance to five or more antimicrobials. This multiresistance was more frequently found among cMLS(B) strains (96.38% MRSA resistant to 6-9 agents).

In vitro activity of cefepime and cefotaxime compared to six other agents against 350 penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae.

From January 1996 to December 1997, we evaluated the in vitro activity of 8 antimicrobials (penicillin, amoxycillin, amoxycillin/clavulanate, cefuroxime, ceftazidime, cefepime, cefotaxime, and imipenem) against 350 Streptococcus pneumoniae clinical isolates collected from two hospitals. Imipenem, cefepime and cefotaxime were the most active antibiotics against penicillin-intermediate (PI) and highly penicillin-resistant (PR) S. pneumoniae with MICs 2- to 8-fold lower than penicillin. Against PI and PR pneumococci amoxycillin and amoxycillin/clavulanate were 2-times less active than cefepime and cefotaxime, while cefuroxime was 4-8-times less active. The majority of strains of serotypes 6B, 23F, 14, 9 and 19 were penicillin-resistant, both intermediate (68%) and highly resistant (32%).

Susceptibility of penicillin-resistant and penicillin-susceptible Streptococcus pneumoniae to newer antimicrobial agents.

Agar dilution minimum inhibitory concentration (MIC) methodology, according to NCCLS guidelines, was used to test the activity of three glycopeptides (LY 333328 [LY], vancomycin [VAN], and teicoplanin [TEI]), four fluoroquinolones (trovafloxacin [TRO], BAY 12-8039 [BAY], ciprofloxacin [CIP], and ofloxacin [OFL]), five macrolide-lincosamide-streptogramin antibiotics (erythromycin [ERY], azithromycin [AZI], miocamycin [MOM], clindamycin CLN], and quinupristin-dalfopristin [SYN] against 126 Streptococcus pneumoniae strains, isolated in Lozano Blesa Hospital of Zaragoza (Spain). MIC50/MIC90 (microg/ml) values for penicillin-susceptible (PS), penicillin-intermediate (PI) and penicillin-resistant (PR) strains show an excellent antipneumococcal activity of LY 333326--a new glycopeptide, for the fluoroquinolones trovafloxacin and moxifloxacin [BAY 12-8039], and for quinupristin/dalfopristin, regardless of the resistance phenotype of the strains.

Resistance to apramycin in two enterobacterial clinical isolates: detection of a 3-N-acetyltransferase IV.

Considering the possible role of farm animals in the contamination of human consumers by plasmid-mediated apramycin-resistant enterobacteria strains, this type of resistance should be tested more systematically in human isolates. Very recently we isolated in Zaragoza one apramycin-resistant Escheria coli strain obtained from the blood of a hospitalized patient; this clinical isolate produced a plasmid-mediated 3-N-aminoglycoside acetyltransferase IV. We describe also the isolation in Madrid of one multiresistant Klebsiella pneumoniae clinical strain. This isolate harbored a single plasmid and carried determinants for apramycin, gentamicin, tobramycin, hygromycin B, streptomycin, and ampicillin, which could be transferred en bloc to E. coli K-12 J62. Extracts from donor and transconjugant strains carrying pUZ6776 plasmid produce acetyltransferase activity AAC(3)-IV and double phosphotransferase activity (HPH and APH(3'')).

A family cluster of giardiasis with variable treatment responses: refractory giardiasis in a family after a trip to India.

Persistence of giardiasis after some of the recommended drugs is occurring with increasing frequency. We describe the follow-up of four members of a family with giardiasis through microscopic observation, immunochromatography and PCRs of tpi and ß-giardin genes. Three patients did not respond to tinidazole but they were cured after quinacrine. However, PCR became negative at 2 months after negativization of stools in two patients and after 1 year in one patient. In all cases Giardia assemblage B was characterized with high homology between all isolates. Further studies are needed to determine the value of PCR in the diagnosis of Giardia infections.

A new pentaplex-nested PCR to detect five pathogenic bacteria in free living amoebae.

Changes in water use and anthropogenic activity have major impacts on the quality of natural aquatic ecosystems, water distribution and wastewater plants. One of the main problems is the presence of some pathogenic microorganisms that are resistant to disinfection procedures when they are hosted by free living amoeba and that in many cases are hardly detectable by culture-based procedures. In this work we report a sensitive, low-cost procedure consisting of a pentaplex-nested PCR that allows simultaneous detection of Legionella pneumophila, Mycobacterium spp., Pseudomonas spp., Vibrio cholerae and the microcystin-producing cyanobacteria Microcystis aeruginosa. The method has been used to detect the presence of these pathogenic bacteria in water and inside free living amoeba. Its validation in 72 samples obtained from different water sources from Aragon (Spain) evidences that Mycobacterium and Pseudomonas spp are prevailing as amoeba-resistant bacteria.