Phenotypic and genotypic characterization of Pseudomonas aeruginosa from cystic fibrosis patients.

Pseudomonas aeruginosa accounts for about one half of all pulmonary infections of cystic fibrosis (CF) patients. In this study, we analyzed 135 P. aeruginosa strains isolated from the expectorations of 55 CF adult patients attending a CF referral center over a period of five years. We assessed the genotype of the strains by pulsed-field gel electrophoresis (PFGE) and analyzed some phenotypic characteristics, such as O serotype, enzyme and mucous production, antibiotics susceptibility, and motility. PFGE allowed the typification of 97.1% of strains, revealing the presence of nine different genomic patterns. The pattern indicated as B was the most frequent, whereas patterns H and I were the most uncommon. Serotyping failed to identify 37.8% of strains and 29 out of 55 patients harbored almost one non-typable (NT) strain. During the five years of the study, we observed a progressive reduction of O6 and O10 types, but an increase of the O1 type and of NT strains. Most strains produced protease, hemolysin, and gelatinase, and were mobile. Several patients harbored the same serotype or genotype in sequential isolates, though characterized by a different susceptibility to antimicrobials. We did not observe a relationship between bacterial genotype and phenotype. This could be due to the fact that PFGE is not sensitive enough to detect subtle genotypic differences. The epidemiological importance of the genotypic characterization of bacteria-colonizing CF subjects and the surveillance measures to be adopted in CF centers are briefly discussed.

Pseudomonas aeruginosa and Burkholderia cenocepacia infections in patients affected by cystic fibrosis: serum resistance and antibody response.

Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic pathogens causing important chronic pulmonary infections in patients affected by cystic fibrosis (CF). The interplay of bacterial and host factors involved in the establishment and evolution of these infections needs further clarification. We investigated the susceptibility of P. aeruginosa and B. cenocepacia derived from CF patients or from the environment to hyperimmune sera obtained from the same CF patients and evaluated the amount of specific antibodies present in these sera. Our data indicate that the bactericidal activity of human serum against these two bacteria is mostly complement-mediated, and that the mucous layer probably confers serum-resistance to B. cenocepacia. The mean amount of antibodies against P. aeruginosa was higher than that against B. cenocepacia. The contribution of these data to the assessment of the importance of the humoral immune response in CF pulmonary infections by Pseudomonas and Burkholderia is briefly discussed.

Evaluation of different compounds as quorum sensing inhibitors in Pseudomonas aeruginosa.

Quorum sensing (QS) controls systems affecting the pathogenicity of many microorganisms; its interruption has an anti-pathogenic effect and can be used in the treatment of bacterial infections. In this study we evaluated QS regulation by Pseudomonas aeruginosa strains and QS inhibition (QSI) by different compounds. The inhibitory activity of 3 macrolide and 3 lincosamide drugs, resveratrol, garlic extract and N-acetylcysteine was tested on 4 P. aeruginosa strains isolated from cystic fibrosis (CF) patients using Chromobacterium violaceum ATCC 12472 as biomonitor. One P. aeruginosa strain, lincomycin and N-acetylcysteine did not show QSI, contrary to other compounds and P. aeruginosa strains. These results indicate that QSI evaluation should be taken into account in the design of new therapeutic strategies to treat P. aeruginosa infections, especially in patients infected by antibiotic-resistant bacteria.

Streptococcus pyogenes collected in Torino (northwest Italy) between 1983 and 1998: survey of macrolide resistance and trend of genotype by RAPD.

We surveyed macrolide resistance in 1,086 isolates of Streptococcus pyogenes, collected between 1983 and 1998, from throat swabs of children with untreated pharyngotonsillitis living in Torino (northwest Italy). In 1983 and 1985, the frequency of erythromycin resistance was 10%, and from 1990 to 1992 it was 4%. However, it rose to 16.6% in 1994 and reached 51% in 1996 before decreasing to 38.5% in 1998. Characterization of the phenotype of resistant isolates revealed the prevalence of constitutive resistance (CR) in 1996, whereas the M phenotype, characterized by resistance to 14- and 15-membered macrolides with susceptibility to clindamycin and streptogramin B, prevailed in 1998. Moreover, in 1997 we observed an increase in the frequency of autoagglutinating bacteria and, in 1998, of OF-negative S. pyogenes. Meanwhile, penicillin tolerance, assessed in the isolates collected from 1990 to 1996, decreased and disappeared. Random amplification of polymorphic DNA (RAPD) was used to obtain the genomic profile of 32 S. pyogenes strains. Four main DNA profiles were demonstrated, generally related to the macrolide-resistance phenotype and for the major part to the T serotype. These results indicate that RAPD is reliable as a first screening method in the epidemiological characterization of resistant S. pyogenes.

Rapid diagnosis of malaria by use of fluorescent probes.

Malaria diagnosis relies on observation of parasites in blood smears and the Giemsa-stained thick blood smear (G-TS) is the reference test. Diagnosis by G-TS in low-density infections requires long periods of observation and experienced microscopists. Examination of Giemsa-stained thin smears enables more reliable differentiation of species but may miss low-grade infections. Fluorescent stains may offer an alternative technique. We compared the Giemsa technique with 4,6-diamidine-2-phenilindolo-propidium iodide (DAPI-PI) stainings in order to evaluate the time required for diagnosis. A Plasmodium falciparum-infected blood specimen was diluted to obtain concentrations ranging from 6192 to 24 parasites/microliters (p/microliter), and thin and thick smears were stained with the two methods. The DAPI-PI proved useful: parasites were easily recognized and their morphology was preserved in thin and thick smears. The method allowed more rapid evaluation of thin smears as compared with Giemsa staining and enabled recognition of parasites in case of low-level parasitemias. The DAPI-PI staining technique may acquire an important role in malaria diagnosis, especially in nonendemic countries where technicians are not experienced with G-TS; in developing countries, it could be used in epidemiologic surveys of populations with low-density parasitemias, for which it enables a fast examination of smears and possibly the identification of parasite species.

Evaluation of the immune response in visceral leishmaniasis.

Detection of parasites in culture or by microscopy is still necessary to make diagnosis of visceral leishmaniasis (VL). Serological methods still need assessment, as they are quick but not very sensitive, especially in immunosuppressed subjects. This paper compares the results obtained with three serological methods (indirect immunofluorescence test (IFAT), direct agglutination test (DAT), and enzyme-linked immunosorbent assay (ELISA) and the specific cell-mediated immune response, evaluated as proliferation and IFN-gamma production by peripheral blood lymphocytes (PBL) following stimulation with heat-killed L. infantum promastigotes. PBL and sera were obtained from 10 healthy donors, 3 VL patients in acute phase, and 3 patients recovering after two glucantim treatment courses. No false positive results were observed with the serological methods. IFAT can be considered the most sensitive and best suited for follow-up, as it allowed a good discrimination between the acute and remission phase. DAT did not discriminate between healthy donors and remission-phase patients, whereas ELISA is unsuited for follow-up, as it did not show any significant difference between remission- and acute-phase patients. Assessment of the cellular response is not recommended for making a diagnosis, because false positive results are frequent. However, a strong cellular response in a patient stands for a successful treatment. IFN-gamma titration is preferable to the proliferation test, because it gives earlier results and does not require the use of radioactive isotopes.

Detection of Pneumocystis carinii by DNA amplification in human immunodeficiency virus-positive patients.

The opportunistic pathogen Pneumocystis carinii (PC) is a frequent cause of a life-threatening pneumonia in human immunodeficiency virus (HIV)-infected individuals and in other immunocompromised hosts. Specimens obtained from 128 bronchoalveolar lavage (BAL) fluid samples from 123 HIV-positive patients with pulmonary disease and undergoing a diagnostic bronchoscopy were evaluated to detect this organism. We have developed a rapid DNA extraction procedure for nested polymerase chain reaction (PCR) using two sets of primers (pAZ102-E, pAZ102-H and P1 = 5'-CTAGGATATAGCTGGTTTTC-3' and P2 = 5'-TCGACTATCTAGCTTATCGC-3'). The results were compared using cytological techniques (direct wet mount, Giemsa, toluidine blue O) and related to the clinical follow-up of patients. The nested PCR had a 91% sensitivity and a 93% specificity. The effect of chemoprophylaxis and the evaluation of the follow-up of patients are discussed. Nested PCR may represent an important additional tool, along with current cytological methods, for the detection of P. carinii; however, at present it cannot replace routine microbiological methods more simple and less expensive.

The effect of subinhibitory concentrations of some antibiotics on the hydrophobicity of gram-negative bacteria.

Cell surface hydrophobicity is currently regarded as an important factor in promoting bacterial adherence to a wide variety of surfaces. This feature was investigated in some Gram-negative bacteria isolated from urinary tract infections and the extent to which their surface characteristics were affected by subinhibitory concentrations of some antibiotics was assayed. Surface properties were evaluated using the salting-out technique (SAT) and bacterial absorption to n-hexadecane (BATH). SAT showed that all except 3 Escherichia coli strains were autoaggregating. BATH detected more hydrophobic characteristics in the stationary phase of bacterial growth. Pretreatment with antibiotics generally reduced hydrophobicity and thus affected the initial reversible phase of attachment of bacteria to eukaryotic cells.

[Recurrent urinary tract infections. Biological suppositions and clinical treatment with thymopentin]. / Infezioni delle vie urinarie recidivanti. Presupposti biologici e risultati clinici del trattamento con timopentina.

BACKGROUND: Urinary tract infections (UTI) are a common usual pathological event. They relapse often due to the periurethral colonization of microorganisms from the intestinal bacterial flora. They also constitute an important and considerable social and clinical problem. The absence of inducing organic conditions or an infective focus at the base of the pathogenetic mechanism suggests the existence of alterations of the immune response of the subject. MATERIALS AND METHODS: In this work we wanted to verify if, in those subjects with relapsing UTI (more than four events every year) cure with "biological" response modifiers, particularly "thymopentin", meaningfully reduced the number of events. RESULTS: The results obtained confirm that for those cases in which the chemo-antibiotic therapy did not have the expected results, it is rational to support it with an immune-modulating drug (thymopentin). In fact the post-therapy reduction of UTI observed during two years of follow-up is statistically significant when compared to the average of UTI before therapy. CONCLUSIONS: The "cost-benefits" analysis should prove to be a saving in favour of the use of thymopentin, taking into consideration the reduction of chemo-antibiotics consumption and the lower number of working hours lost every year.

[Protozoan infection (Blastocystis hominis) concomitant with Pseudomonas sp. peritonitis in continuous ambulatory peritoneal dialysis (CAPD)]. / Infezione protozoaria (Blastocystis hominis) concomitante a peritonite da Pseudomanas sp. in corso di dialisi peritoneale ambulatoriale continua (CAPD).

Case-report of protozoal infection (Blastocystis bominis) during Pseudomonas peritonitis in male patient with intestinal diverticulosis on continuous ambulatory peritoneal dialysis (CAPD) treatment for chronic renal failure (CRF). Microscopic morphology and cultural characteristics are summarized from current literature. Photographic images in phase contrast from fresh-observation of faeces and peritoneal fluid are reported. Although other Protozoa (e.g. Acanthamoeba free-living) have already been found in dialysis fluid, this is the first case, referred in literature, of Blastocystis bominis infection in CAPD patients. Some pathogenetic hypothesis are done involving Blastocystis bominis in peritoneal infection, especially in immunodepressed patients like dialysed ones. Although many chemotherapeutics are provided for this protozoiasis during enteritis, in our case no supplement was required except specific antibiotic therapy for Pseudomonas infection. Symbion or pathogen? Is now-a-day the question which troubles parasitologists. Systemic research of Protozoa in dialysed patients is anyhow advisable.

Proliferative response of human T cells to stimulation with Leishmania infantum and L. major promastigotes in logarithmic and stationary growth phase.

T cells from healthy donors, non-exposed to Leishmania, proliferative and produce lymphokines when cultured in the presence of killed Leishmania promastigotes. We compared the stimulative activity of L. major and L. infantum promastigotes harvested in logarithmic and stationary growth phase on human T cells by performing a proliferation kinetics during the first 5 days of culture. Stationary-phase promastigotes induced a good response at the 4th-5th day, whereas the effect of logarithmic-phase promastigotes was almost undetectable. Both species gave a similar pattern. The implications of these findings in the current model of immunologic reaction to leishmaniasis are discussed.

A one-year survey of respiratory and urinary pathogens and their antimicrobial susceptibility.

A one-year (1993) survey of the distribution of pathogens causing respiratory and urinary infections and their antimicrobial susceptibility was performed. The most common bacteria isolated from the lower respiratory tract of patients in a district general hospital were Pseudomonas aeruginosa (35.9%) and Staphylococcus aureus (21.4%). About half of the Pseudomonas strains revealed a resistance to imipenem and gentamicin, whereas almost all Staphylococcus strains were resistant to penicillin G. The most common isolates from urine of in and out-patients were Escherichia coli (32.3% and 39.8%) and Enterococcus faecalis (16.6% and 14.2%). Escherichia coli strains were largely susceptible to almost all chemoantibiotics tested, whereas Enterococcus faecalis demonstrated a high resistance pattern. Pseudomonas aeruginosa isolated from urine were more sensitive to chemoantibiotics than respiratory strains and the susceptibility of Staphylococcus aureus isolated from hospitalized or out-patients was different. A periodic monitoring system devised to give information about the circulation of bacteria and the chemoantibiotic resistance in a local context would be useful to assess the local trends and select drugs for therapy.

Human PBMC proliferative response to Leishmania infantum promastigotes is inhibited by anti-IFN-gamma mAb gamma 123.

PBMC from individuals both exposed and non-exposed to leishmaniae proliferative and produce interferon-gamma (IFN-gamma) following stimulation with Leishmania antigens. We studied the kinetics of the proliferative response of PBMC from non-exposed individuals and from patients recovering from visceral leishmaniasis due to Leishmania infantum, using heat-killed stationary-phase promastigotes of L. infantum as stimulating agent. The kinetics of both groups followed a similar temporal pattern, with higher values in the patient's group. Moreover, we observed that in both groups the activation was dose-dependently inhibited following the addition of gamma 123 anti-IFN-gamma monoclonal antibody. These results indicate the need for IFN-gamma in the activation process of PBMC induced by Leishmania antigens and stress the role of IFN-gamma in the immune response to leishmaniasis. The relevance of the elucidation of the immune response mechanism in human leishmaniasis for therapy and vaccination is briefly discussed.

Protective role of the pefloxacin-IFN-gamma association in Leishmania major-infected mice.

Four groups of female Balb/c mice were inoculated in the left hind footpad with 30 microliters of RPMI 1640 medium containing 10(7) Leishmania major amastigotes/ml. One group was injected sc with 200 microliters of RPMI 1640 containing 180 micrograms of pefloxacin for 20 days, a second group with the same amount of medium containing 100 units of recombinant murine interferon gamma (rmIFN-gamma). The third group was treated with the association, while the fourth group received plain medium in an identical regimen. Pefloxacin or IFN-gamma significantly decreased the size of primary lesions, while their association was significantly more efficient in this respect, in reducing the incidence of metastatic lesions, and in clearing parasites from the spleen. We also investigated the effect of pefloxacin on the activation of mouse spleen cells by Concanavalin A (Con A) in vitro, without detecting any interference on the proliferative response or IFN-gamma production.

[Human sparganosis. A clinical case]. / Sparganosi umana. Caso clinico.

After reviewing the international literature, the Authors report an uncommon case of sparganosis localized in a submandibular gland.

New chemotherapeutic strategies against malaria, leishmaniasis and trypanosomiases.

Due to the persistent lack of suitable vaccines, chemotherapy remains the only option for the treatment of patients infected by protozoan parasites. However, most available antiparasitic drugs have serious disadvantages, ranging from high cost and poor compliance to high toxicity and rapid induction of resistance. In recent decades basic research laboratories identified a considerable number of promising new molecules, but their development has not been pursued in depth by pharmaceutical firms because of poor prospects of economic return. The establishment of adequately funded public-private partnerships is currently reversing the trend. This review deals with new drugs against Plasmodium, Leishmania and Trypanosoma parasites, focusing on the molecules that are in the most advanced stage of development. The purpose of this article is to provide the reader with a panoramic view of the updated literature on the challenges and strategies of contemporary antiprotozoal drug research, paying the due attention to the already published reviews.

New antimicrobial frontiers.

New antimicrobials able to counteract bacterial resistance are needed to maintain the control of infectious diseases. The last 40 years have seen the systematic tailoring and refinement of previously identified antibiotics, to produce a multitude of semi-synthetic derivatives that share their mechanism of action with the original molecules. The major limit of this approach is the emergence of multi- and cross-resistant bacterial strains, favoured by the selective pressure inherent to the targeting of specific enzymes. The most promising new strategies aim to the development of molecules that, targeting essential bacterial structures instead of specific enzymatic activities, achieve infection control without enforcing a selective pressure on bacteria. This review, based on the consultation of the up-to-date literature, deals with antimicrobial peptides and some antivirulence factors.