Variation of the antimicrobial susceptibility profiles of Burkholderia cepacia complex clonal isolates obtained from chronically infected cystic fibrosis patients: a five-year survey in the major Portuguese treatment center.

The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia complex (Bcc) bacteria requires extensive and aggressive antibiotics therapy, exposing these bacteria to prolonged antibiotics-selective pressure. In the present study, we have compared the susceptibility patterns to 13 antimicrobials of 94 Bcc isolates obtained from 15 Portuguese CF patients in the course of chronic infection during a five-year survey. These isolates were previously genotyped and represent 11 different strains of the species B. cenocepacia (subgroups A and B), B. cepacia, B. multivorans, and B. stabilis. The results are consistent with the notion that CF Bcc isolates are resistant to the most clinically relevant antimicrobials and suggest an uneven distribution of resistance rates among the different species, with B. cenocepacia subgroup A isolates being the most resistant. Phenotypic variants exhibiting differences in the antimicrobial susceptibility patterns were obtained from the sputum samples of clinically deteriorated CF patients during chronic lung infection. The isolation of resistant variants coincided with periods of pulmonary exacerbation and antibiotics therapy.

Screening of auramine-stained smears of all fecal samples is a rapid and inexpensive way to increase the detection of coccidial infections.

INTRODUCTION: Coccidia are important causes of diarrhea that is often indistinguishable from other forms of community-acquired diarrhea. However, the detection of oocysts is often only performed when explicitly requested, as part of the ova and parasite (O&P) examination. Reappraisal and understanding of the accurate staining characteristics of auramine O (AuO), which stains nucleic acids, may permit the inexpensive and reliable identification of coccidian oocysts at routine workup of all fecal samples. METHODS: AuO-stained smears were prepared from all stool samples received for stool culture in transport medium (SC) and from concentrated stools received for the ova and parasite (O&P) examination. RESULTS: A total of 3732 samples for stool cultures and 3132 samples for O&P examinations were included. Ninety-one samples (1.3%) from 52 patients yielded Coccidia (45 Cryptosporidium spp and 7 Isospora belli). In seven cases oocysts were only detected in samples sent for stool culture in transport medium. The oocysts showed a typical staining pattern and were easy to recognize. The observation of one smear took only around 30seconds, and the reagents and glass slide for one smear did not exceed US$ 0.03. CONCLUSIONS: The screening of all fecal samples with AuO-stained smears is a rapid and inexpensive way to increase the detection of coccidial infections, which in most laboratories can be incorporated into the microscopic workup for mycobacteria.

Group A Streptococci from carriage and disease in Portugal: evolution of antimicrobial resistance and T antigenic types during 2000-2002.

In this study, we analyzed the antimicrobial resistance properties and T antigenic types of 511 isolates collected in Lisbon district, Portugal, from throat swabs of healthy subjects (n=341), during 2000-2002 and from diverse infection sites (n=170) of outpatients and inpatients, during 1999-2002. Erythromycin resistance was higher in tonsillitis/pharyngitis (27.4%) and skin infection isolates (21.1%), than in carriage and invasive isolates (

Growth of Mycobacterium tuberculosis in conventional BacT/ALERT FA blood culture bottles allows reliable diagnosis of Mycobacteremia.

The conventional BacT/ALERT FA blood cultures supported the ample growth of Mycobacterium tuberculosis in seeding experiments and appeared to perform as reliably as the BACTEC Myco/F-Lytic vials in the recovery of M. tuberculosis from blood in HIV-infected patients. Overall, blood cultures were positive in 39% of patients with tuberculosis.

Streptococcus agalactiae in a large Portuguese teaching hospital: antimicrobial susceptibility, serotype distribution, and clonal analysis of macrolide-resistant isolates.

Group B streptococci are emerging as a cause of serious infection worldwide. The capsular polysaccharides are not only important virulence factors but also the target of vaccine development efforts. Serotypes III (24.6%), V (23.4%), Ia (17.8%), and II (16.3%) were the most prevalent among 252 Streptococcus agalactiae isolates collected during 1999-2002 in the largest hospital of Lisbon, Portugal. The substantial proportion of bacteremic patients (17 neonates and 21 adults) in this period illustrates the present importance of S. agalactiae as a cause of invasive disease. All isolates were fully susceptible to penicillin (MIC(50) = 0.064 microg/ml; MIC(90) = 0.094 microg/ml, range 0.008-0.094), cefotaxime, chloramphenicol, ofloxacin, and vancomycin. Resistance was found to tetracycline (75.4%), erythromycin (10.7%), and clindamycin (9.9%). Of the 27 erythromycin-resistant isolates, 70.4% had the cMLS(B), 22.2% the iMLS(B), and 7.4% the M phenotype. All isolates presenting the M phenotype carried the mef(A) gene, whereas the erm(B) gene was found in a large fraction of MLS(B) isolates (n = 17) and only a small proportion (n = 7) the erm(A) gene [erm(TR) variant]. All isolates carried a single macrolide-resistance determinant. Macrolide resistance was not attributable to a single clone as evidenced by distinct serotype and pulsed-field gel electrophoretic profiles. Careful surveillance of S. agalactiae invasive infections in Portugal is essential, and the treatment or intrapartum prophylaxis of patients who are allergic to penicillin should be guided by contemporary resistance patterns observed in the country.

Case of aortic endocarditis caused by Lactobacillus casei.

A case of Lactobacillus aortic valve endocarditis in a 53-year-old immunocompetent patient with past history of rheumatic fever is reported. Clinical symptoms began after a dental extraction and the patient's diet included several yogurts per day. Blood, bone marrow cultures and the replaced aortic valve were positive for Lactobacillus: The clinical isolate was identified as Lactobacillus casei by 16S rDNA sequencing.

Molecular characterization of a new class 3 integron in Klebsiella pneumoniae.

Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum beta-lactamase was encoded by bla(GES-1), previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the bla(GES-1) gene cassette was located on a new class 3 integron. The integron was 2863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The bla(GES-1) gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between bla(OXA-10)-type and aac(6')-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the bla(GES-1) gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.

Assessment of drinking water contamination at springs along the road to Santiago (NW Spain).

With the aim of assessing the potability of drinking water, 31 different springs sited along the road to Santiago "The French Way" in Galicia (NW Spain) were studied. Twenty-one parameters, according to Spanish Legislation have been determined by Official Methods. It was observed that the parameters: pH, nitrate, ammonia, total coliforms, fecal coliforms and fecal streptococcus did not always comply with the levels established by Spanish Legislation. The bacteriological characters were the most restrictive of the sanitary quality of drinking water, only 19% of the springs were drinkable during all the study. According to the Factorial Analysis in Principal Component applied to the obtained results, it was concluded that the parameters: conductivity, hardness, dry residue, nitrate and fecal coliforms can be very indicative for assessing water sanitary quality.

Spontaneous bacterial peritonitis in patients with hepatic cirrhosis: evaluation of a treatment protocol at specialized units.

INTRODUCTION: Spontaneous bacterial peritonitis is a common and severe complication in patients with cirrhosis and ascitis. Its prognosis clearly depends on its precocious clinical recognition and efficacious therapy. AIM: To optimize a treatment protocol, after auditing clinical efficacy and describe microorganisms implicated at our institution. MATERIAL AND METHODS: Retrospective study of clinical files of patients with hepatic cirrhosis with positive culture of ascitic fluid (AF) and/or an AF polymorphonuclear (PMN) count of more than 250/mm3, treated at our units between 1st January, 2000 and 31st December, 2001 (n = 38). Patients showed a median age of 49 years (30-76), 63% of which were male. Forty-eight percent were classified as belonging to Child-Pugh B class, and 52% to C. RESULTS: First, considering cases with PMN > 250/mm3 (n = 29), antibiotics were given to all patients (cefotaxime and ampiciline). Fifty-two percent had hepatic encephalopathy, 42% had fever, 66% abdominal pain. In 42% a microorganism was isolated. Although 24% of fatal cases (only two related to infection), we noted a 73% clinical and laboratorial response. Five patients (72%) that died, showed renal failure by the time of death. Second, in all cases with positive culture of ascitic fluid (n = 21), 42% of which with PMN > 250/mm3 and 9 monobacterial nonneutrocytic bacterascites' cases, one only agent was found: E. coli in 36%, Streptococci (37%), Staphylococci (14%), and other (14%): Klebsiella oxytoca, n = 1; Salmonella enteritidis, n = 1; Enterococcus faecium, n = 1, Acinectobacter anitratus, n = 1. Only one of the agents, E. faecium (3%) showed in vitro sensitivity exclusively to ampiciline; all other were cefotaxime sensitivite. CONCLUSIONS: Our protocol will be modified, to treat patients with spontaneous bacterial peritonitis with cefotaxime, as monotherapy. Albumin infusion will also be added to the protocol, as, we found renal failure to be an important negative prognosis factor.

Peritonitis bacteriana espontánea en pacientes con cirrosis hepática: evaluación de un protocolo de tratamiento en unidades especializadas / Spontaneous bacterial peritonitis in patients with hepatic cirrhosis: evaluation of a treatment protocol at specia lized units

Introducción: la peritonitis bacteriana espontánea es una complicación frecuente y grave en los pacientes con cirrosis y ascitis. Su pronóstico depende claramente de su reconocimiento clínico precoz y de que el tratamiento sea eficaz. Objetivo: optimizar un protocolo de tratamiento, después de auditar su eficacia clínica, y describir los microorganismos implicados en nuestra institución. Material y métodos: estudio retrospectivo de las historias clínicas de los pacientes con cirrosis hepática y cultivo positivo de líquido ascítico (LA), y / o un recuento de polimorfonucleares (PMN) en LA de más de 250/mm3, tratados en nuestras unidades entre el 1 de enero de 2000 y el 31 de diciembre de 2001 (n = 38). Los pacientes presentaban una mediana de edad de 49 años (30-76), siendo varones el 63 por ciento de ellos. El 48 por ciento se clasificaron como pertenecientes a la categoría Child-Pugh B, y el 52 por ciento a la C. Resultados: en primer lugar, considerando los casos con PMN > 250/mm3 (n = 29), se administraron antibióticos a todos los pacientes (cefotaxima y ampicilina). El 52 por ciento tenían encefalopatía hepática, el 42 por ciento fiebre y el 66 por ciento dolor abdominal. En el 42 por ciento se aisló un microorganismo. Aunque el 24 por ciento de los casos fueron fatales (sólo 2 en relación con infecciones), observamos un 73 por ciento de respuestas clínicas y analíticas. Cinco de los pacientes (72 por ciento) que fallecieron presentaban insuficiencia renal en el momento de morir. En segundo lugar, en todos los casos con cultivo positivo de líquido ascítico (n= 21), el 42 por ciento de los cuales presentaban PMN > 250 mm3, con nueve casos de ascitis monobacteriana no neutrocítica, sólo se halló un agente: E. coli en el 36 por ciento, estreptococos (37 por ciento), estafilococos (14 por ciento) y otros, como Klebsiella oxytoca, n = 1; Salmonella enteritidis, n = 1; Enterococcus faecium, n = 1, y Acinectobacter anitratus, n = 1. Sólo uno de los agentes, E. faecium (3 por ciento) mostró sensibilidad exclusivamente a la ampicilina in vitro. Todos los demás fueron sensibles a la cefotaxima. Conclusiones: nuestro protocolo se modificará para tratar con cefotaxima en monoterapia a los pacientes con peritonitis bacteriana espontánea. También se añadirán al protocolo las infusiones de albúmina, ya que encontramos que la insuficiencia renal suponía un importante factor pronóstico negativo. (AU)

Feasibility of introducing rejection criteria for stool cultures in a teaching hospital in Portugal.

The possible introduction of rejection criteria for stool cultures (

Survey of Klebsiella pneumoniae producing extended-spectrum beta-lactamases at a Portuguese hospital: TEM-10 as the endemic enzyme.

One hundred and thirty-eight isolates of Klebsiella pneumoniae showing resistance to ceftazidime were isolated from different wards of the Hospital de Santa Maria, Lisbon. The genomic DNA of the isolates was analysed by pulsed-field gel electrophoresis (PFGE) and two patterns were predominant. In all isolates the presence of a single large plasmid of about 50 kb suggested that propagation of the outbreak prominently involved plasmid spread. The deduced amino acid sequence indicated the presence of a TEM-10 beta-lactamase. This extended-spectrum beta-lactamase was present among K. pneumoniae isolates, was widely disseminated in different wards and remained persistent as a result of an outbreak involving the dissemination of both the multi-resistance plasmids harbouring the bla gene and the isolates themselves.

Molecular typing and exopolysaccharide biosynthesis of Burkholderia cepacia isolates from a Portuguese cystic fibrosis center.

This work describes the first epidemiological survey of Burkholderia cepacia involved in pulmonary infections among the Portuguese population with cystic fibrosis (CF) who attended the major CF treatment Center in Lisbon at Sta. Maria Hospital from 1995 to the end of 1997. The characterization of the genomic relatedness of the isolates was based on the analysis of their ribopatterns (with EcoRI) followed by construction of a ribotype-based phylogenetic tree. This study was complemented with macrorestriction fragment analysis by pulsed-field gel electrophoresis. After optimization of the solid growth medium, we found that exopolysaccharide (EPS) production by B. cepacia CF isolates is not as rare a phenomenon as was thought before; indeed, 70% of the isolates examined were EPS producers.

Accumulation of mercury in edible macrofungi: influence of some factors.

The levels of mercury in terrestrial ecosystems are generally very low, but the strong accumulation in some species of macrofungi is an exception from this rule. Mercury contents of 41 samples of edible mushrooms fruiting bodies representing eight species (six wild and two cultivated) were determined by an anodic stripping voltammetric technique (ASV) using a gold disc as the working electrode. Wild fungi were collected in unpolluted and polluted areas in the Province of Lugo (NW Spain). Influence of some factors (ecology, species, traffic pollution, and morphological portion) and the importance of mushrooms as a dietary source of mercury have been studied. Wild saprophytic species showed higher levels (ppm DW) than mycorrhizals species, with the exception of Boletus pinicola. There were significant differences according to the species analyzed (p < 0.001), the highest average content of mercury was found in Boletus pinicola (7.37 ppm DW), and the range was 0.35-33.07 ppm DW for hymenophore and 0.18-20.30 ppm DW for the rest of the fruit body. The cultivated species accumulated lower than wild species because the mean life is shorter. The traffic pollution factor did not show significant differences, so mushrooms are not realiable bioindicators of traffic pollution by mercury. Hymenophore was always the morphological portion that contained the highest mercury levels (p < 0.05), and the mean ratio of hymenophore/rest of the fruit body was 2.13. The mercury concentrations were compared to literature data and levels set by legislation, and the contribution of mushrooms to the weekly intake of mercury per person was evaluated. The possible health risk for people is pointed out.