Association between antimicrobial resistance and biofilm forming ability of Salmonella enterica serotypes from commercial broiler farms in Brazil.

1. This study determined the antimicrobial resistance profile and the biofilm-forming ability of Salmonella enterica strains isolated from commercial broiler houses over a three-year period in southern Brazil.2. Of the 720 drag swabs analysed, 37 (5%) tested positive for non-typhoidal Salmonella spp. and S. Heidelberg was the most frequent serovar.3. Among the antimicrobial resistant strains (83.8%; 31/37), resistance was most common to tetracycline, ampicillin and nalidixic acid. Multidrug resistance was found in 65% (24/37) of the isolates, with a large proportion of multidrug resistant S. Heidelberg strains (81%; 13/16).4. In total, 65% (24/37) of the isolates showed the ability to produce biofilm and multiple antimicrobial resistance was negatively correlated with biofilm formation.5. Strains susceptible to all tested antimicrobials tended to form stronger biofilms than multidrug resistant ones. This suggested that Salmonella spp. with less antimicrobial resistance depend more on the protection provided by biofilm to survive in the farm environment.

Caution at choosing a particular colony-forming unit from faecal Escherichia coli: it may not represent the sample profile.

Data about phylogenetic classification of Escherichia coli colonizing calves, lambs and foals are routinely neglected and restricted to outdated methodologies, even in the context of antimicrobial susceptibility (AS) testing. Thus, the aim of this study was to determine the phylogenetic diversity and the AS profile of E. coli colony-forming units (CFUs) from faecal samples of healthy animals. Five CFUs of E. coli were randomly selected from each faecal culture of calves (n = 13), foals (n = 13) and lambs (n = 13), totalizing 195 CFUs phylo-typed by quadruplex PCR. The AS profile of five CFUs from 15 samples (five from each animal species; n = 75 isolates) against nine drugs was determined by agar diffusion test. We found E. coli belonging to all phylo-groups already described, except D group, with the predominance of B1 (65% CFUs; 126/195) in the three-animal species sampled. Most faecal samples of calves (77%; 10/13) and foals (69%; 9/13) harboured both pathogenic and nonpathogenic E. coli. All faecal samples showed CFUs with diverse AS profile, highlighting the ineffectiveness of tetracycline, sulphonamide and ampicillin. As a key point, our data reinforce the importance to select at least four E. coli CFUs for AS testing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides relevant data about the high phylogenetic and antimicrobial susceptibility diversity observed in Escherichia coli colony-forming units (CFUs) from a bacteriological culture of faeces from healthy calves, foals and lambs. The selection pressure exerted by the herd treatment may directly impact the intestinal microflora of animals that have never been treated. Finally, we emphasize the importance of Clinical Laboratory Standards Institute guidelines and we recommended to analyse at least four E. coli CFUs to determine, in particular, the antimicrobial susceptibility profile of faecal isolates, independent of the animal's health status.

Antibacterial potential of phytochemicals alone or in combination with antimicrobials against fish pathogenic bacteria.

AIMS: This study investigated the antibacterial activity of five phytochemicals (carvacrol, citral, eugenol, linalool and thymol) alone or in combination with florfenicol or oxytetracycline against bacteria isolated from silver catfish (Rhamdia quelen). We also analysed the potential of these compounds to inhibit biofilm formation and haemolysis caused by the bacteria. METHODS AND RESULTS: Bacteria were tested with antimicrobials to calculate the multiple antibiotic resistances. The checkerboard assay was used to evaluate a putative synergy between five phytochemicals and antimicrobials against the strains isolated. The biofilm formation inhibition assay was performed with phytochemicals and antimicrobials, and the haemolysis inhibition assay was performed with the phytochemicals. Carvacrol, eugenol and thymol were the most effective phytochemicals. Three combinations (linalool with florfenicol or oxytetracycline against Aeromonas hydrophila and citral with oxytetracycline against Citrobacter freundii) demonstrated synergy in the checkerboard assay. All phytochemicals inhibited biofilm formation and haemolysis activity. CONCLUSION: The tested phytochemicals showed satisfactory activity against fish pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The phytochemicals did not present antagonistic interactions with the antimicrobials, allowing their combined use, which may contribute to a decrease in the use of conventional drugs and their residues in aquatic environment.

Neutrophil chemotaxis and enzyme release competitive inhibition by a diazoacetamide pepsin inhibitor.

Treatment of human neutrophils with a reagent (diazoacetylnorleucine methyl ester plus copper ion) which covalently labels the active site of the acid proteases, pepsin and cathepsin D, inhibits neutrophil chemotaxis and enzyme release stimulated by the chemoattractants pepstatin and formylmethiony peptides. In contrast, chemotaxis and enzyme release in response to zymosan activated serum are not affected. Furthermore, diazoacetylnorleucine methy ester plus copper competes with [3H]formylmethionyl leucylphenylalanine for binding to neutrophils. Since pepstatin shares binding sites with formylmethionyl leucylphenylalanine, the present data suggest that diazoacetylnorleucine methyl ester plus copper reacts with the neutrophil receptor for pepstatin and formylmethionyl peptides, and thus may be useful in further characterization of this structure.

A critical assessment of the prognostic value of HIV-1 RNA levels and CD4+ cell counts in HIV-infected patients. The Swiss HIV Cohort Study.

OBJECTIVE: To determine to what extent human immunodeficiency type 1 (HIV-1) RNA levels and CD4+ cell counts predict clinical outcomes in a general HIV-1-infected population. METHODS: Community-based prospective study (Swiss HIV Cohort Study) including 394 HIV-1-infected patients, randomly selected from 4 strata of CD4+ cell counts (0 to < 0.05, 0.05 to < 0.20, 0.20 to < 0.50, and > or = 0.50 x 10(9)/L). Levels of HIV-1 RNA, CD4+ cell counts, and other variables were evaluated from samples collected between 1991 and 1993 for their ability to predict death and clinical progression. RESULTS: Patients were followed up on average for 29 months. Baseline HIV-1 RNA levels, CD4+ cell counts, clinical stage, and beta 2-microglobulin levels independently predicted survival, whereas only HIV-1 RNA levels and CD4+ cell counts independently predicted clinical progression. Multivariate relative hazards (RHs) for death ranged from 1.0 to 5.4 across quartiles of CD4+ counts, but only from 1.0 to 1.8 across quartiles of HIV-1 RNA. For clinical progression, gradients of risk were similar for CD4+ counts (1.0-4.2) and for HIV-1 RNA (1.0-3.1). In patients with CD4+ cell counts less than 0.05 x 10(9)L, HIV-1 RNA levels predicted neither death nor clinical progression. Finally, the number of HIV-1 RNA copies per CD4+ cell was the best predictor of death (multivariate RH, 1.0-9.7 across quartiles) and clinical progression (multivariate RH, 1.0-4.1). CONCLUSIONS: Levels of HIV-1 RNA and CD4+ cell counts provided independent and complementary information on clinical outcomes. The RNA/CD4+ ratio was the best single predictor. In patients who had fewer than 0.05 x 10(9)/L CD4+ cells, HIV-1 RNA levels had little prognostic value.

HAART in HIV-infected patients: restoration of antigen-specific CD4 T-cell responses in vitro is correlated with CD4 memory T-cell reconstitution, whereas improvement in delayed type hypersensitivity is related to a decrease in viraemia.

OBJECTIVE: To analyse prospectively the effect of highly active antiretroviral treatment (HAART) on CD4 T-cell responses in vitro and in vivo in HIV-infected patients. DESIGN: Prospective study with 49 protease inhibitor-naive adult patients. Data were collected at baseline and after 3 and 6 months of HAART. METHODS: In vitro CD4 T-cell reactivity was analysed by stimulation of peripheral blood mononuclear cells with several antigens. In vivo CD4 T-cell reactivity (delayed type hypersensitivity) was assessed by Multitest Merieux. Both measurements were correlated to CD4 (memory) T-cell count and HIV-1 viraemia. RESULTS: Restoration of specific CD4 T-cell proliferation was observed in most patients. The in vitro T-cell response was restored more frequently against antigens to which the immune system is constantly exposed (Candida albicans, Mycobacterium tuberculosis, M. avium) as compared with a low-exposure antigen (tetanus toxoid). Overall, delayed type hypersensitivity detection rate increased under HAART. Multivariate analysis showed improvement of antigen-specific T-cell proliferation to be significantly associated with an increase in memory CD4 T-cells, whereas improvement of the delayed type hypersensitivity response was associated with a decrease in plasma HIV-1 RNA. CONCLUSIONS: HAART for 6 months restored antigen-specific CD4 T-cell response to several antigens. In vitro immune reconstitution was closely correlated with an increase in memory CD4 cells. Restoration of delayed type hypersensitivity was associated with suppression of viraemia. It appears that in addition to expansion of memory CD4 cells, suppression of viraemia following HAART may allow an improved inflammatory reaction, thus providing even stronger immune reconstitution.

Community-acquired pneumonia. A prospective outpatient study.

We initiated a prospective study with a group of practitioners to assess the etiology, clinical presentation, and outcome of community-acquired pneumonia in patients diagnosed in the outpatient setting. All patients with signs and symptoms suggestive of pneumonia and an infiltrate on chest X-ray underwent an extensive standard workup and were followed over 4 weeks. Over a 4-year period, 184 patients were eligible, of whom 170 (age range, 15-96 yr; median, 43 yr) were included and analyzed. In 78 (46%), no etiologic agent could be demonstrated. In the remaining 92 patients, 107 etiologic agents were implicated: 43 were due to "pyogenic" bacteria (39 Streptococcus pneumoniae, 3 Haemophilus spp., 1 Streptococcus spp.), 39 were due to "atypical" bacteria (24 Mycoplasma pneumoniae, 9 Chlamydia pneumoniae, 4 Coxiella burnetii, 2 Legionella spp.), and 25 were due to viruses (20 influenza viruses and 5 other respiratory viruses). There were only a few statistically significant clinical differences between the different etiologic categories (higher age and comorbidities in viral or in episodes of undetermined etiology, higher neutrophil counts in "pyogenic" episodes, more frequent bilateral and interstitial infiltrates in viral episodes). There were 2 deaths, both in patients with advanced age (83 and 86 years old), and several comorbidities. Only 14 patients (8.2%) required hospitalization. In 6 patients (3.4%), the pneumonia episode uncovered a local neoplasia. This study shows that most cases of community-acquired pneumonia have a favorable outcome and can be successfully managed in an outpatient setting. Moreover, in the absence of rapid and reliable clinical or laboratory tests to establish a definite etiologic diagnosis at presentation, the spectrum of the etiologic agents suggest that initial antibiotic therapy should cover both S. pneumoniae and atypical bacteria, as well as possible influenza viruses during the epidemic season.

Evaluation of 2-SP transport medium for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by two automated amplification systems and culture for chlamydia.

AIMS: To assess the performance of 2-sucrose-phosphate based transport medium (2-SP) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by an automated commercial polymerase chain reaction (PCR) and ligase chain reaction (LCR) compared to centrifugation culture on McCoy cells for C trachomatis. Second, to compare both amplification systems for initial diagnostic testing of a low prevalence population for sexually transmitted diseases. METHODS: Four hundred and eighty one consecutive urogenital and conjunctival specimens were examined. All tests were performed on the same specimen collected with a dacron swab and transported in 2-SP medium. Samples that were positive by culture or by both PCR and LCR were considered to be true positives. RESULTS: The prevalences of C trachomatis and of N gonorrhoeae were 2.7% and 0.4%, respectively. PCR had a resolved sensitivity and specificity of 100% and 99.8%, respectively, for C trachomatis, and 100% and 98.9%, respectively, for N gonorrhoeae. LCR was 100% sensitive and specific for both pathogens. The resolved sensitivity of the shell vial assay was 85%. No culture positive sample would have been missed by PCR or LCR. The inhibition rate for PCR was 4.8%. CONCLUSIONS: 2-SP medium proved to be suitable for both PCR and LCR. It is not limited to any one test manufacturer and allows the performance of amplification techniques and viral and chlamydia culture from the same specimen. The LCR was more reliable than PCR on initial testing. However, hands on time is longer, and no amplification control is available for LCR.

Detection of mumps virus in clinical specimens by rapid centrifugation culture and conventional tube cell culture.

Conventional tube cell culture was compared with a 2 day and 5 day spin-amplified shell vial indirect immunofluorescence assay for the detection of mumps virus in swabs from the area of Stensen's duct. The sensitivity and specificity of the shell vial assay were 95.9 and 100%, respectively. The shell vial detected 66.3% of the positive cultures within 2 days of inoculation while the first positive results were available by conventional tube cell culture after 3 days (1.6%) reaching 72.4% of all culture positive specimens after 7 days. These data suggest that a centrifugation shell vial indirect immunofluorescence assay may be useful for rapid detection of mumps virus in clinical specimens.

IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins.

Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (p52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (pp150, aa 994-1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELISA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection.

Locked-in state in Borrelia burgdorferi meningitis.

The case is reported of a 28-year-old woman with persistent tetraplegia following acute meningitis due to Borrelia burgdorferi infection. The patient developed erythema chronicum migrans before radicular pain occurred in the upper extremities. The poor clinical outcome was suggestive of pontine infarction due to vasculitis of branches of the basilar artery.

Chronic progressive neurological involvement in Borrelia burgdorferi infection.

Five patients with chronic meningitis were hospitalized several times for progressive neurological symptoms. The clinical manifestations included cranial neuritis, radiculoneuritis, myelitis and encephalitis. In two cases cerebral infarction occurred. The course was commonly characterized by a tendency to deteriorate. From the clinical point of view, it was repeatedly difficult to exclude multiple sclerosis or tuberculous meningitis. Finally, specific antibodies against Borrelia burgdorferi were detected by indirect immunofluorescence assay. The diagnosis of a borreliosis was not considered initially because there was no history of tick-bite or erythema chronicum migrans, and the neurological involvement of the central nervous system seemed unusual. The latency between the first symptoms and diagnosis varied from 3 months to 5 years. After a parenteral, high-dose therapy with penicillin, there was a significant improvement in all patients. In two cases, there was evidence of intrathecally produced antibodies to myelin basic protein.

The immune response to S. aureus in atopic dermatitis.

The skin of patients with atopic dermatitis is heavily colonized with S. aureus, and their immune response to S. aureus shows some particular features: (1) A selective hyporesponsiveness to purified S. aureus cell walls in delayed type hypersensitivity skin reactions. (2) The presence of IgE to cell walls and soluble antigens of S. aureus in patients with high serum IgE levels. (3) Elevated cell wall IgE do not correlate with positive immediate skin reactions to whole S. aureus and their cell walls. (4) Regional lymphadenopathy but not impetiginization is associated with high total IgE and S. aureus cell wall IgE. We suggest that these changes in the immune response to S. aureus are related to the chronic S. aureus colonization of the skin.

[Angiosarcoma of the kidney. Case report and review of the literature]. / Angiosarkom der Niere. Fallbeispiel und Literaturübersicht.

The primary renal angiosarcoma is a rarely seen highly malignant tumor. Making a diagnosis based on histology may prove difficult. Because of hematogenous formation of metastases and bad prognosis in most cases we recommend that the surgical intervention be followed by the well tolerated systemic chemotherapy with Doxorubicin and Ifosfamid.

[HIV diagnosis 1998]. / HIV-Diagnostik 1998.

Due to considerable technical progress during the last few years the diagnosis of HIV-infection has been substantially improved. Third generation antibody screening assays, which also detect antibodies of the IgM and IgA type, have considerably narrowed the immunological window. The determination of the viral load in peripheral blood employing nucleic acid amplification techniques is now generally available and used for diagnostic and prognostic purposes as well as for the monitoring of antiviral therapy. To detect a HIV-infection the antibody screening assay is primarily used and complemented by the HIV-1 p24 antigen assay provided an early primary infection is suspected. In the latter case the antibody screening assay is often negative or indeterminate, while the p24 antigen assay is positive. According to the 1998 guidelines of the Federal Office for Public Health, the physician will be informed of the screening assay result without the need to await a confirmatory test in case of a reactive screening assay in the first sample. Confirmation, e.g. by immunoblot, will be done in a second blood sample which should be sent to the laboratory as soon as possible. EDTA-blood is recommended for this purpose, because it is best suited for quantification of plasma viremia, which has become a prerequisite for the institution and follow-up of antiretroviral treatment. The second sample will also serve to exclude false positive results due to clerical errors, and to determine the type of HIV, i.e. HIV-1 or HIV-2. The concept outlined should accelerate the availability of reactive test results to the physician and should reduce the cost of the diagnostic procedure.