Haloferax volcanii for biotechnology applications: challenges, current state and perspectives.

Haloferax volcanii is an obligate halophilic archaeon with its origin in the Dead Sea. Simple laboratory culture conditions and a wide range of genetic tools have made it a model organism for studying haloarchaeal cell biology. Halophilic enzymes of potential interest to biotechnology have opened up the application of this organism in biocatalysis, bioremediation, nanobiotechnology, bioplastics and the biofuel industry. Functionally active halophilic proteins can be easily expressed in a halophilic environment, and an extensive genetic toolkit with options for regulated protein overexpression has allowed the purification of biotechnologically important enzymes from different halophiles in H. volcanii. However, corrosion mediated damage caused to stainless-steel bioreactors by high salt concentrations and a tendency to form biofilms when cultured in high volume are some of the challenges of applying H. volcanii in biotechnology. The ability to employ expressed active proteins in immobilized cells within a porous biocompatible matrix offers new avenues for exploiting H. volcanii in biotechnology. This review critically evaluates the various application potentials, challenges and toolkits available for using this extreme halophilic organism in biotechnology.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems.

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.

Epilepsy, cysticercosis, and toxocariasis: a population-based case-control study in rural Bolivia.

OBJECTIVE: To assess the relationship between epilepsy and infection with Taenia solium and Toxocara canis with a case-control study, in the rural area of the Cordillera Province, Bolivia. METHODS: A preliminary two-phase door-to-door prevalence survey determined the prevalence of epilepsy and identified cases and control subjects. At least two control subjects per case were selected, matching on sex, age, and community of residence. Cases and control subjects were assessed serologically for antibodies against T. canis by ELISA and against T. solium by enzyme-linked immunoelectrotransfer blot (EITB). RESULTS: The prevalence survey found 130 confirmed cases of epilepsy, of which 113 were eligible for the case-control study (59 partial seizures and 54 generalized seizures). Two hundred thirty-three control subjects were selected. Multivariable analysis for a matched case-control study was carried out. There was an association between EITB positivity for T. solium and epilepsy with an OR of 1.85 (95% CI 0.99 to 3.4) for all cases. A stronger association was found in those with partial epilepsy with a late onset of disease (15 years and older), where the OR was 3.66 (95% CI 1.10 to 12.10). A positive association was also found with T. canis for all cases with an OR of 2.70 (95% CI 1.41 to 5.19). This increased for those with late-onset partial epilepsy to an OR of 18.22 (95% CI 2.10 to 158.10). CONCLUSION: This finding suggests that both neurocysticercosis and toxocariasis may in part explain the higher prevalence of epilepsy, particularly partial epilepsy, in developing countries.

Prevalence of epilepsy in rural Bolivia: a door-to-door survey.

OBJECTIVE: To carry out a door-to-door survey in rural areas of the Cordillera Province, Santa Cruz Department, Bolivia, to determine the prevalence of neurologic diseases (epilepsy, stroke, parkinsonism, and peripheral neuropathy) in a sample of approximately 10,000 inhabitants. METHODS: A team of nondoctor health workers administered a standard screening instrument for neurologic diseases-a slightly modified version of the World Health Organization protocol. All subjects found positive during the screening underwent a neurologic examination. RESULTS: On screening, the authors found 1,130 positive subjects, of whom 1,027 were then investigated by neurologists. On the basis of the definition proposed by the International League Against Epilepsy, we detected 124 epileptic patients (prevalence, 12.3/1,000), 112 of whom had active epilepsy (prevalence, 11.1/1,000) on the prevalence day (November 1, 1994). Peak age-specific prevalence occurred in the 15 to 24-year age group (20.4/1,000). Sex-specific prevalence was higher in women (13.1/1,000) than men (11.4/1,000). Eighty-nine patients (71.8%) underwent a standard EEG recording. Considering both EEG and clinical data, partial seizures were the most common type (53.2%) based on the classification of the International League Against Epilepsy. The mean age at onset was 20.7 years for partial seizures and 13.6 years for generalized seizures. Only 10.5% of patients had received specific treatment for more than 2 months of their life. CONCLUSION: This report on epilepsy prevalence in Bolivia confirms that epilepsy is a major health problem in rural areas of developing countries.

In-vitro differentiation of human monocytes into mature macrophages during long-term cultures.

A method is described which allows human peripheral blood monocytes from any given donor to differentiate in vitro into mature macrophages. About 90% of the starting monocytes are maintained during the long-term culture and are matured to macrophages. Thus cell loss is minimal and the resulting population of mature macrophages can be regarded as representative for all possible macrophage subpopulations present in peripheral blood. These cultures represent a standardized model for in-vitro studies on the role of mature macrophages in various immunological reactions.

Is Streptococcus pneumoniae a nosocomially acquired pathogen?

Streptococcus pneumoniae is most prominently a major cause of community-acquired infections of the respiratory tract, central nervous system, and bloodstream, but there is an increasing interest in its role in the epidemiology of hospital-acquired infections. Penicillin-resistant pneumococcal strains appeared 3 decades ago and now are present worldwide, often displaying multiple resistance due to antibiotic selective pressure. Horizontal spread can cause either sporadic cases or hospital outbreaks, primarily in younger children and elderly patients. Pneumococcal transmission from one patient to another can be documented by polymerase chain reaction or pulsed-field gel electrophoresis typing. Nosocomial acquisition of infection, along with pediatric age, previous hospitalization, and previous beta-lactam therapy, are the main risk factors significantly associated with penicillin-resistant pneumococcal infections. Nosocomial acquisition also is associated with higher mortality from pneumococcal disease. The importance of penicillin resistance as a risk factor significantly associated with higher mortality from pneumococcal infection is found in some studies, but not in others. Mortality from pneumococcal pneumonia is approximately the same for human immunodeficiency virus (HIV)-infected patients without acquired immunodeficiency syndrome (AIDS) as for HIV-negative subjects, but it is significantly higher in AIDS patients. Penicillin-resistant strains are involved in the vast majority of hospital outbreaks, whether presenting as clinically manifest infection or a simple colonization. Pneumococcal vaccination is recommended universally in order to lower the incidence of invasive infection, although a number of problems can limit its effectiveness.

Infections in multiple myeloma.

Multiple myeloma is a relatively rare but severe hematologic malignancy. Marked depression in production of normal immunoglobulins, mild neutropenia, and alkylant/steroid therapy or BMT/SCT all produce major suppression of the immune system in the totality of patients. Recurrent bacterial, fungal, and viral infections are an important cause of morbidity and the most common cause of death in these subjects. Prompt diagnosis and appropriate anti-infective chemotherapy are essential in order to reduce the risk of mortality.

Streptococcus pneumoniae as an agent of nosocomial infection: treatment in the era of penicillin-resistant strains.

Streptococcus pneumoniae is a well-known agent of community-acquired infections such as sinusitis, otitis media, pneumonia, bacterial meningitis, bacteremia and acute exacerbations of chronic bronchitis. However, the role of S. pneumoniae as a cause of nosocomial infections of respiratory tract, bloodstream and central nervous system is more and more recognized, primarily in high-risk patients with depression of their immune function. Therapy of pneumococcal infections is made difficult by the emergence and spread of bacterial resistance to penicillin and other beta-lactams as well as to a number of antimicrobials such as macrolides, chloramphenicol, tetracyclines and sulfonamides. This epidemiological situation is a cause for concern world-wide, but it primarily affects some European countries, North America, South Africa and the Far East. The main consequence on therapeutic grounds is that in severe infections such as bacterial meningitis, the addition of vancomycin to a third-generation cephalosporin is advisable while awaiting laboratory test results, even in areas with low prevalence of penicillin-resistant pneumococci. However, a beta-lactam agent can also be a valid choice in the presence of potentially lethal infections such as pneumonia or in the case of penicillin intermediately resistant isolates. In recent years, new alternative molecules have been introduced into clinical practice for therapy of infections caused by penicillin-resistant pneumococci. In both in vivo and in vitro studies, drugs of the classes of fluoroquinolones (levofloxacin, moxifloxacin, gatifloxacin), streptogramins (quinupristin/dalfopristin) and oxazolidinones (linezolid) have shown good microbiologic and clinical efficacy against penicillin-resistant pneumococci. In this era of world-wide spread of penicillin-resistant pneumococci, use of polysaccaride or conjugated vaccines is highly recommended.

Evaluation of a rapid immunochromatographic test for the serologic diagnosis of tuberculosis in Italy.

OBJECTIVE: To determine specificity, sensitivity and predictive values of a rapid immunochromatographic assay (ICT tuberculosis) for the diagnosis of tuberculosis (TB) in an Italian clinical setting, and to identify tentative new guidance for the interpretation of test results. METHODS: The ICT tuberculosis test is an immunochromatographic test based on the detection of IgG antibodies directed against five highly purified antigens secreted by Mycobacterium tuberculosis during active growth. Sera from 60 patients with active pulmonary (48 sputum smear-positive and six sputum smear-negative cases) and extrapulmonary (six cases) TB were obtained. Personal, anamnestic and clinical data were investigated and recorded for each patient. The control groups comprised 156 subjects: 40 healthy individuals, half of them Mycobacterium bovis BCG-vaccinated, and 116 patients with mycobacterial diseases other than TB (five cases), with nonmycobacterial lung diseases (30 cases), with nonmycobacterial nonlung diseases (30 cases), with nonmycobacterial diseases and rheumatoid factors positivity (30 cases), and with asymptomatic HIV infection (21 cases). For 21 individuals the test was simultaneously performed with both serum and whole blood sample. Each positive result of the ICT test was reported with regard to the number (1-4), position (A, B, C, D) and color intensity (+ to ++++) of the evidenced lines in order to assess the quality of the antibody response. RESULTS: The overall sensitivity and specificity were 56.7% and 90.4%, respectively. The sensitivity for pulmonary TB patients was 61.1% (66.7% for smear-positive and 16.7% for smear-negative cases) and 16.7% for extrapulmonary TB patients. The difference between ICT results in pulmonary TB patients and control subjects was statistically significant (P < 0.0001). The analysis of the positive ICT tests revealed that samples with strong color intensity (>/=++) and specific antibodies bound to antigens immobilized on line D were significantly more frequent in TB patients than in controls (P = 0.001 and P= 0.027, respectively). ICT test results with the presence of at least three visible lines were more often observed in the TB patients than in controls, although not reaching statistical significance (P = 0.052). No difference was observed between the results of the ICT test performed both on serum and whole blood sample. CONCLUSIONS: The ICT tuberculosis test was confirmed to be rapid and easy to perform without requiring special equipment, both on serum and whole blood sample. Our data, in accordance with those obtained in a previous study conducted in extra-European countries, confirmed higher sensitivities for the smear-positive TB patients than for the smear-negative TB patients, and for pulmonary TB patients than for the extrapulmonary TB patients. Data obtained on the quality of antibody response in the ICT positive samples, might be used to improve the performance of the test.

Nosocomial bacterial and fungal meningitis in children; an eight year national survey reporting 101 cases. Pediatric Nosocomial Meningitis Study Group.

One hundred and one cases of nosocomial meningitis in children from a national survey over 8 years have been analyzed for risk factors and outcome. From 101 cases, 115 organisms were isolated. Seventy six were Gram-positive bacteria, 29 were Gram-negative and there were ten fungal isolates. Major risk factors for acquisition of nosocomial meningitis were neurosurgery (70.2%), ventriculoperitoneal shunt (42.9%), prior therapy with broad spectrum antibiotics (64.1%), central venous catheter (94.5%), premature neonates with very low birth weight (32.8%) and total parenteral nutrition (68.8%). Overall attributable mortality was 14. 9%; in bacterial infection it was 13.2% and in fungal nosocomial meningitis, 30.0%. Higher mortality was significantly related to perinatal pathology with CNS abnormality, prematurity polymicrobial infection with Enterobacteriaceae and concomitant bacteraemia. Prematurity in neonates, very low birth weight and infection with Enterobacteriaceae were significantly associated with a worse outcome.

Clinical and microbiologic efficacy and safety profile of linezolid, a new oxazolidinone antibiotic.

Gram-positive cocci are important causes of infection both in the community and in the hospital, with repercussions on mortality and increased economic costs. Treatment of these infections is made difficult by the increasing emergence of multi-resistant organisms, primarily among Gram-positive cocci, such as vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci, and penicillin-resistant pneumococci. Linezolid, a member of the new class of synthetic antimicrobials named oxazolidinones, has several favourable characteristics including high activity against multiresistant Gram-positive cocci. In a number of clinical trials, linezolid showed good clinical and microbiologic efficacy in the therapy of infections caused by these organisms. It can be considered a valid option for treating both community- and hospital-acquired infections due to multiresistant Gram-positive cocci.

Distribution and antibiotic resistance of isolates from lower respiratory tract and blood cultures from patients in three Italian intensive care units: a 2-year comparison.

The distribution and antibiotic resistance of major pathogens isolated from patients in ICUs were studied by three Italian microbiological laboratories. Consecutive aerobic strains were collected over two different time periods from protected brushing bronchoscopy, broncho-alveolar lavage and blood cultures. A total of 420 strains were isolated during the first period (47.3% gram-negative and 52.7% gram-positive) and 412 over the second period (50.5% gram-negative and 49.5% gram-positive). Pseudomonas aeruginosa was the most frequently isolated organism from the respiratory tract followed by Staphylococcus aureus. Methicillin resistance was 47.9 and 44.5% in S. aureus and 63.0 and 65.1% in coagulase-negative staphylococci over the two periods. No glycopeptide-resistance was found in gram-positive organisms. Ceftazidime-resistance in Klebsiella pneumoniae was very high.

Antistaphylococcal (MSSA, MRSA, MSSE, MRSE) antibiotics.

S. aureus and coagulase-negative staphylococci such as S. epidermidis are important causes of infection of the bloodstream, cardiac valves, implanted devices, and skin, with repercussions on mortality and increased economic costs. Treatment of staphylococcal infections is made difficult by the increasing emergence of resistance to beta-lactams and other antimicrobials, including reduced susceptibility to glycopeptides. Penicillin must be used for infrequent penicillin-susceptible isolates, oxacillin and nafcillin are to be considered the major option for penicillin-resistant staphylococci, and glycopeptides are the drugs of choice for infections caused by methicillin-resistant strains. Co-trimoxazole, lincosamides, macrolides, tetracyclines, and fluoroquinolones are alternative agents, primarily in subjects allergic to beta-lactams. Newly introduced or experimental drugs, such as streptogramins (quinupristin-dalfopristin), oxazolidinones (linezolid), carbapenems (LY 333328), everninomicins (SCH 27899), and derivatives of tetracyclines (glycylcyclines), could be useful for therapy of infections caused by multiresistant staphylococci.

Lumbar pain in a married couple who likes cheese: brucella strikes again!

A 69-year-old man living in Florence reported fever and acute lumbar pain one month after transurethral resection of a superficial transitional cell carcinoma of the bladder. The radionuclide bone scan suggested metastatic lesions of the L3-L4 vertebrae. However cobalt treatment was ineffective. A bone biopsy of L4 showed an inflammatory pattern and antibiotic therapy was started which did not produce any clinical improvement. Six months after the onset of the back pain brucellar spondylitis was serologically diagnosed and treatment with doxycycline and streptomycin produced a significant clinical and radiological improvement. After 2 months the patient's wife presented with fever and lumbar pain, and brucellar spondylitis was diagnosed as well. An extensive epidemiological examination revealed that 8 months earlier the family had eaten unpasteurized goat cheese and serological examination of the entire family showed that 3 out of 4 members had significant titres of brucellar antibodies. Finally it was discovered that 4 months after consuming the cheese the third infected subject experienced an episode of epidydimoorchitis for which no diagnosis and effective treatment was found. This family cluster of brucellar infection indicates that a high degree of suspicion in the diagnosis of brucellosis is necessary even in non-endemic areas, to reduce the delay in the diagnosis and treatment of the disease and to prevent the occurrence of complications that may prove difficult to treat.

Which prophylactic regimen for which surgical procedure?

For optimal prevention of infection subsequent to a surgical intervention, it is necessary to follow a series of general principles, including the classification of the type of surgical intervention, the characteristics of the antibiotic used, and the route and the time of its administration. Moreover, with reference to the different types of surgery, other factors assume importance: the etiology of the infection and the ability of the antibiotic to achieve adequate levels in the tissues at the beginning of the infective process. In general abdominal, biliary, and obstetric-gynecologic surgery, which covers many clean-contaminated and contaminated interventions for which antibiotic prophylaxis has been shown to be the most effective, the etiology is often mixed (aerobic and anaerobic flora) with a predominance of gram-negative microorganisms. Thus, an appropriate prophylactic regimen must consider a third-generation cephalosporin, such as cefotaxime, that is effective against most gram-negative bacteria, in particular against Klebsiella pneumoniae. Acylureido penicillins can also be used because of their activity against enterococci, gram-positive microorganisms that are also causes of infection in this area of surgical intervention. Combining an antimicrobial such as clindamycin or metronidazole, which are particularly active against anaerobes, may be recommended as well. In urologic surgery, most infections are caused by Enterobacteriaceae; in addition to the antimicrobial spectrum, the ability of the antibiotic to concentrate adequately in the urine and renal tissue must also be considered. Beta-lactam antibiotics are the agents of choice, in particular, third-generation cephalosporins, aztreonam, and acylureido penicillins. In cardiac, orthopedic, and partially in neurologic surgery, where most infections are due to gram-positive bacteria (primarily methicillin-resistant staphylococci), antibiotic prophylaxis should include a glycopeptide agent (teicoplanin, vancomycin). In the field of surgical prophylaxis, more experience has been accumulated with cefotaxime, used as a short-course regimen or as a convenient single dose, than with any other newer cephalosporin. Cefotaxime's broad spectrum of action provides coverage against most potential pathogens and, when used as a single dose, is both convenient and cost-effective.

Urosepsis in the critical care unit.

Critical care unit patients show a higher risk of developing a bloodstream infection than ward patients. The urinary tract is the main source of hospital-acquired secondary bloodstream infection. Nosocomial urinary tract infection is promoted by bladder catheterization in the vast majority of cases. Aerobic gram-negative bacilli are the prevalent agents of bloodstream infection secondary to a nosocomial urinary tract infection. Sepsis and septic shock are severe complications of these infections in the critical care patient. Management of patients with a septic process of urinary source calls for the combination of adequate life-supporting care, an appropriate antibiotic therapy, and innovative adjunctive measures. Accurate catheter care is the best measure to adopt for the prevention of urosepsis.

Once-daily dosing regimen for aminoglycoside plus betalactam combination therapy of serious lower respiratory tract infections.

Aminoglycosides are important antibacterial agents for treatment of serious gram-negative bacillary infections including lower respiratory tract infection. Once-daily aminoglycosides result in higher peak and lower trough plasma concentrations than conventional multiple daily dosing regimens; once-daily aminoglycoside therapy is equally effective, generally less toxic and much less expensive and therefore this regimen is more and more frequently used for treatment of suspected or confirmed gram-negative bacillary infections and of febrile episodes in neutropenic patients, in particular in combination with an appropriate betalactam antibiotic. Despite the lack of studies on this topic, once-daily aminoglycosides in combination with a betalactam agent can be used in subjects with lower respiratory tract infection, including patients with cystic fibrosis, in which tobramycin appears to be the aminoglycoside antibiotic of choice.

[Pathogenetic mechanisms responsible for producing a secondary immunodeficiency state]. / Meccaanismi patogenetici responsabili della produzione di uno stato di immunodeficienza secondaria.

Abnormalities of the immune response can be secondary to old age, to several pathologic conditions (i.e. diabetes mellitus, renal failure, solid and lymphohematologic neoplasias, leukopenia, malnutrition, autoimmune diseases, AIDS), to surgical stress or to burns, and to immunosuppressive therapies, both medical (corticosteroids, cytotoxic agents, antilymphocytic globulins) and surgical (splenectomy) as well as radiant (extensive radiotherapy). Old age can affect both humoral (reduced antibody synthesis) and cell-mediated (thymus involution, diminished ratio Th/Ts, depression of both delayed hypersensitivity reactions and cytotoxic activity of K cells) immune response. Hyponutrition, often observed in the elderly, adds a reduced production of secretory IgA, lysozyme and interferon, diminished complementary activity, phagocytosis defects, and vitamin deficits. Furthermore, in some chronic diseases we can observe reduced primary antibody response or depression of delayed hypersensitivity reactions (renal failure, neoplasias), changes in leukocyte functions (diabetes mellitus, leukemias and lymphomas) and, in particular in solid neoplasias, increased activity of Ts lymphocytes and the presence of circulating immunocomplexes. Changes in phagocytosis, opsonization and chemotaxis are typically seen in burns, whereas surgical stress can cause some inhibition of cell-mediated immunity. Finally, after splenectomy it is possible to observe an increased synthesis of IgA and IgG and, on the contrary, reduced production of IgM and properdin.

Effect of enoxacin, norfloxacin and pefloxacin on polymorphonuclear leukocytes chemiluminescence.

We studied the interactions between the neutrophilic granulocytes and three quinolone antibiotics, enoxacin, norfloxacin and pefloxacin, using the luminol-enhanced chemiluminescence method to evaluate the respiratory burst of the cells. Neutrophils obtained from healthy human volunteers were exposed to different therapeutic concentrations of the drugs for one and three hours and then opzonized zymosan was added to stimulate chemiluminescence reaction. None of the three drugs tested exercised any influence on neutrophil function.

Influence of teicoplanin on the chemiluminescence of phagocytosing human granulocytes.

Teicoplanin is a new glycopeptide antibiotic that is structurally related to vancomycin. It is especially active against virtually all gram positive bacteria including methicillin-resistant staphylococci. The aim of this study was to evaluate the influence of teicoplanin on the phagocytosis of human neutrophils, using the chemiluminescence reaction method. We incubated three different concentrations (2, 5, 10 microg/ml) of teicoplanin for one and three hours with polymorphonuclear leukocytes of healthy human volunteers and then measured the chemiluminescence reaction during zymosan phagocytosis. The neutrophil function was not influenced by teicoplanin.