Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy / Caracterización enzimática de aislamientos clínicos y ambientales de Cryptococcus neoformans de Italia

Background. Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. Aims. In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. Methods. Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. Results. Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. Conclusions. The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too (AU)

Antecedentes. Cryptococcus neoformans es una levadura encapsulada que produce infecciones oportunistas. Los factores de virulencia involucrados en la patogénesis de la criptococosis incluyen la existencia y el tamaño de la cápsula polisacarídica, la producción de melanina por medio de la enzima fenoloxidasa, el crecimiento a 37°C y la secreción de ciertas enzimas como proteinasa, fosfolipasa y ureasa. Existen otras enzimas que son secretadas por C. neoformans, pero su papel en la virulencia de este hongo aún no es conocido. Objetivos. Se investigó la producción de fosfolipasa tanto en aislamientos de C. neoformans obtenidos de pacientes como de aislamientos recuperados de deposiciones de aves, y se comparó el grado de producción con el de la síntesis de la enzima fenoloxidasa. Además, distingue las cepas mediante la definición de la actividad de 19 enzimas extracelulares diferentes. Métodos. Se estudiaron 205 aislamientos clínicos de C. neoformans y 32 ambientales. La producción de fenoloxidasa se determinó por el crecimiento de colonias de color marrón en medio de Staib. Para determinar la actividad fosfolipasa extracelular se utilizó el método semicuantitativo en placa con yema de huevo. Con el método comercial API ZYM se determinó la producción de otras 19 enzimas extracelulares. Resultados. El análisis estadístico de los resultados mostró una producción de fosfolipasa significativamente mayor entre los aislamientos clínicos en comparación con los ambientales. No se encontraron diferencias significativas entre ambos grupos en la producción de fenoloxidasa. En lo referente a las 19 enzimas extracelulares valoradas mediante el sistema API ZYM, la fosfatasa ácida mostró la mayor actividad en ambos grupos. Respecto a la enzima α-glucosidasa, nuevamente los aislamientos clínicos presentaron una actividad significativamente mayor. Todos los aislamientos de ambos grupos presentaron actividad leucina-arilamidasa, si bien los aislamientos clínicos procesaron mayor cantidad de sustrato de manera significativa. Conclusiones. La mayor producción de enzima fosfolipasa entre los aislamientos clínicos evidencia que esta enzima puede estar implicada en la patogénesis de la criptococosis. También es interesante la actividad extracelular de las enzimas fosfatasa ácida, α-glucosidasa y leucina-arilamidasa, valorada por medio del sistema comercial API ZYM. Diversos estudios apuntan a que estas enzimas están implicadas en la virulencia de bacterias y parásitos; nuestros resultados muestran también su posible implicación como factores de virulencia en las infecciones por Cryptococcus (AU)

Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy.

BACKGROUND: Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. AIMS: In order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. METHODS: Two hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. RESULTS: Statistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. CONCLUSIONS: The higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too.

[Methicillin and vancomycin-resistant staphylococci and vancomycin-resistant enterococci in a hematology ward]. / Diffusione di stafilococchi meticillino e vancomicino-resistenti ed enterococchi vancomicino-resistenti in un reparto ospedaliero di ematologia.

Microbiological sampling of surfaces and air was performed in a hematology department, to monitor the presence of methicillin and vancomycin-resistant staphylococci and of vancomycin-resistant enterococci. The hematology department was divided in two areas, one of which with controlled access.Staphylococci were detected equally in the two areas while vancomycin-resistant enterococci were isolated only occasionally. The results show that the implementation of strict protocols for accessing hospital wards is not justified if effective cleaning and disinfection practices are not adopted.

Extracellular phospholipase activity of Malassezia strains isolated from individuals with and without dermatological disease.

BACKGROUND: The Malassezia genus includes mainly lipophilic yeasts belonging to the cutaneous microbiota of man and other mammals. Some Malassezia species have been associated with various dermatological diseases. The factors permitting the transformation of yeasts of the Malassezia genus from a commensal organism to a pathogenic agent are still little known but the production of various enzymes such as lipase, phospholipase and lipoxygenase could contribute to the pathogenic activity of these yeasts. AIMS: Here we have determined and compared the extracellular phospholipase activity of sixty human isolates of Malassezia so as to relate this feature to the species of Malassezia and to the origin (from dermatological diseases or not) of the strains examined. METHODS: Phospholipase production was determined using the semi-quantitative egg-yolk plate method. RESULTS AND CONCLUSIONS: Malassezia obtusa, Malassezia slooffiae, Malassezia globosa, Malassezia restricta had difficulty developing in the chosen culture medium so that it was not possible to measure phospholipasic activity. Malassezia pachydermatis showed the highest phospholipase activity. Twenty-nine Malassezia sympodialis strains produced phospholipase; the isolates from patients with pityriasis versicolor had significantly higher phospholipasic activity than those isolated from healthy individuals. This observation suggests that the phospholipasic activity of Malassezia may play a role in the onset of skin lesions, especially in the case of pityriasis versicolor.

Invasive pulmonary aspergillosis in neutropenic patients and the influence of hospital renovation.

To evaluate the effects of airborne Aspergillus contamination during and after the renovation work of a Florentine haematology unit, we conducted (November 2003-January 2005) a strict programme of environmental fungal surveillance. Air samples were taken from patients' rooms, along the corridors inside the wards, along the corridor between wards and outside the building. The concentration of Aspergillus fumigatus was high along the corridor between the two haematology wards (2.98 CFU m(-3)), lower in the non-neutropenic patients' rooms and outside the hospital building (1.53 and 1.42 CFU m(-3), respectively), very low in the neutropenic patients' rooms (0.09 CFU m(-3)). During this period, three proven cases (A. fumigatus), two probable ones and two possible cases of invasive pulmonary aspergillosis were documented in 97 patients with acute leukaemia (7%). The three cases of proven aspergillosis coincided with the period of renovation work and with the period in which we have found the maximum concentration of A. fumigatus along the corridor. These data suggest a possible relationship between environmental fungal contamination and the incidence of invasive aspergillosis, and underline the importance of environmental surveillance.

Management of antifungal susceptibility testing in Italy: comparative results of 2 nationwide surveys (1999 and 2004) in 102 Italian hospitals.

The purpose of this study was to verify the standard procedures and minimum level of knowledge of Italian public laboratories involved in the management of antifungal susceptibility testing (AST). Two nationwide surveys were performed in 1999 and 2004. One hundred and two Italian hospitals located in 85 provincial capitals (82.5%) participated to these surveys. In 1999, 28 (27.5%) laboratories versus 16 (15.7%) in 2004 stated that they did not perform any susceptibility testing. Some discrepancies observed in the survey confirm that AST is difficult to be correctly managed, and that it can be performed only in very well-trained centers. The great variability of the results of MIC determination and clinical interpretation underlines the urgent need to improve knowledge about indications, method choice, and interpretative criteria for AST both for clinical microbiologists and clinicians.

Isolation of Trichosporon in a hematology ward.

During mycologic monitoring of the air in a hematology ward, we found massive air contamination caused by Trichosporon asahii, both in the room where neutropenic patients were staying and the corridor immediately outside the room. This fungal species had never been isolated in previous samplings. The urine culture taken from one of the patients in this room, whose urinary catheter had been removed immediately prior to air sampling, resulted positive for T. asahii. Both macroscopic and microscopic morphologic observation was insufficient for confirming the hypothesis of a close relationship between the strains isolated from the patient, from the air in the room and corridor. Therefore, we used genomic typing with random amplified polymorphic DNA (RAPD). The five primers used, (GTG)(5), (GACA)(4), M13, OPE01, RC08, produced different patterns of polymerase chain reaction (PCR) products; the genomic profiles obtained with the same primer, however, resulted perfectly superimposable for all the strains. This result led us to conclude that the massive air contamination caused by T. asahii can have effectively been determined by the removal of the urinary catheter from the patient who presented an asymptomatic infection caused by this microorganism.

Two years of a fungal aerobiocontamination survey in a Florentine haematology ward.

The control of microbial air contamination in hospital wards has assumed great importance particularly for those hospital infections where an airborne infection route is hypothesised, such as aspergillosis. Invasive aspergillosis represents one of the most serious complications in immunocompromised patients. For some authors there is a direct association between this pathology and the concentrations of Aspergillus conidia in the air; in addition, reports of aspergillosis concurring during building construction have been frequent. In this study, two haematology wards were monitored for about 2 years in order to make both a qualitative and quantitative evaluation of fungal burden in the air, also in relation to major construction and demolition work taking place in the same building. Air samples were taken from the hospital rooms of neutropenic patients, in the corridors of their ward and outside the building. Total fungal concentration resulted higher outside (mean 572 Colony Forming Units/m3 of air), lower in the corridors (147 CFU/m3) and even lower in the rooms (50 CFU/m3). In all the samples we found the development of at least one fungal colony. Cladosporium was the most frequently isolated genus (57%), in contrast to Aspergillus spp. (2%). The average concentration of Cladosporium spp. was 24 CFU/m3 in the rooms, 78 CFU/m3 in the corridors and 318 CFU/m3 outside. The average concentration of Aspergillus spp. was 1.2 CFU/m3 in the rooms, 3.5 CFU/m3 in the corridors, 5.6 CFU/m3 outside. Our observations show low concentrations of Aspergillus fumigatus and A. flavus in all the environments examined and particularly in the rooms (0.09 and 0.10 CFU/m3 respectively); this observation could explain the absence of cases of invasive aspergillosis during the period of air monitoring in the two haematology wards.

Multicenter evaluation of an enzyme immunoassay (Platelia Aspergillus) for the detection of Aspergillus antigen in serum.

Invasive aspergillosis is a serious problem for immunocompromised patients, especially if neutropenic. The diagnosis of this infection is complicated, since clinical symptoms are often similar to those of other fungal diseases. The chance of detecting the presence of a specific antigen in the serum could confirm the suspected clinical diagnosis and. perhaps, be useful for the follow-up of the patient. The Medical Mycology Committee of the Associazione Microbiologi Clinici Italiani (AMCLI) decided to evaluate in a multicenter prospective study (from I November 1998 to 28 February 1999) the performance of the Platelia Aspergillus Kit (Bio-Rad) for the detection of Aspergillus galactomannan in human serum. The enrolled patients included various groups of immunosuppressed patients (mostly neutropenic). Blood samples were drawn at the time of enrollment. This decision was based upon a clinical diagnosis of probable aspergillosis (antibiotic non-responsive fever for at least 96 hours, cough, hemophthosis and positive chest X-ray). Additional blood samples were drawn on days 3, 6, 9, 12, 15 and 21. Culture and histopathologic examinations were performed according to the individual laboratory workflow. For each patient the laboratory filled a form with all the available clinical information, to create a database on which to evaluate the results of the test. During the study, 187 patients with various kinds of immunosuppression were enrolled. A total of 256 sera were tested: for 117 patients (62.6%) only the basal sample was tested, whereas for the 70 symptomatic patients (37.4%) multiple specimens (range: 1-6) were tested. The results allowed the laboratories to exclude (68.6%) or confirm (31.5%: confirmed and/or probable) the clinical diagnosis of invasive aspergillosis; 4 cases remained undetermined. Based on the results of this study, it seems that the use of this test should be limited to those patients with clinical symptoms of aspergillosis.