1.09 to 5.10; P ‫؍‬ 0.03), infection with overall biofilm-forming Candida species (OR, 2.33; 95% CI, 1.26 to 4.30; P ‫؍‬ 0.007), and Acute Physiology and Chronic Health Evaluation III scores (OR, 1.03; 95% CI, 1.01 to 1.15; P < 0.001) as independent predictors of mortality. Notably, if mortality was analyzed according to the different biofilm-forming Candida species studied, only infections caused by C. albicans (P < 0.001) and C. parapsilosis (P ‫؍‬ 0.003) correlated with increased mortality. Together with well-established factors, Candida biofilm production was therefore shown to be associated with greater mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.

We report a case of fatal pulmonary infection caused by Mycobacterium abscessus in a young patient with cystic fibrosis, who underwent bipulmonary transplantation after a 1-year history of severe lung disease. Fifteen days after surgery he developed septic fever with progressive deterioration in lung function. M. abscessus, initially isolated from a pleural fluid specimen, was then recovered from repeated blood samples, suggesting a disseminated nature of the mycobacterial disease. Drug susceptibility testing assay, performed on two sequential isolates of the microorganism, showed a pattern of multidrug resistance. Despite aggressive therapy with several antimycobacterial drugs, including clarithromycin, the infection persisted, and the patient died. CASE REPORTA 20-year-old man with cystic fibrosis (CF) underwent bilateral pulmonary transplantation in July of 1999. For approximately 1 year prior to admission he had been suffering from severe pulmonary disease with intermittent episodes of septic fever, and during this period he had been hospitalized several times. Sputum cultures had yielded Pseudomonas aeruginosa and Aspergillus fumigatus, whereas blood cultures (collected either via catheter and by venipuncture) were positive for Saccharomyces cerevisiae. On the basis of these microbiological findings, he had received several courses of antibiotic and antifungal therapy consisting of quinolones, third-generation cephalosporins, and amphotericin B, which had, however, produced only temporary remission of the symptoms. All cultures for mycobacteria (blood, sputum, urine, etc.) were repeatedly negative; sputum culture was contaminated by P. aeruginosa.Bilateral lung transplantation was performed on 20 July 1999. There were no intraoperative complications, and the patient was started on immunosuppressive therapy.On 5 August 1999, the patient developed a septic syndrome, and computed tomography (CT) performed on 7 August 1999 revealed a hyperdense lesion in the right paracardiac region with features of parenchymal involvement and a reactive pleural effusion. In light of the previous fungal recovery from clinical specimens, he was promptly started on intravenous amphotericin B therapy. On 6 August 1999 the pleural fluid was cultured for mycobacteria and was inoculated into Middlebrook 7H12 medium (BACTEC 12B; Becton Dickinson Diagnostic Instruments, Sparks, Md.) and Löwenstein-Jensen slant (BioMerieux, Marcy l'Etoile, France). On 8 August 1999, sputum and blood samples were also submitted for mycobacterial studies. A smear of respiratory samples showed many acid-fast bacilli.All three specimens yielded a rapidly growing mycobacterium identified as Mycobacterium chelonae and subsequently as M. abscessus, and on 20 August 1999 the patient was started on intravenous ciprofloxacin, amikacin, and clarithromycin. Sensitivity studies soon revealed, however, that the isolates were clearly resistant to all three drugs, as well as to all of the others tested (Table 1). In fact, after 1 month of antibiotic therapy the patient...

SummaryOur previous investigation on Candida glabrata azole-resistant isolates identified two isolates with unaltered expression of CgCDR1/CgCDR2, but with upregulation of another ATP-binding cassette transporter, CgSNQ2, which is a gene highly similar to ScSNQ2 from Saccharomyces cerevisiae. One of the two isolates (BPY55) was used here to elucidate this phenomenon. Disruption of CgSNQ2 in BPY55 decreased azole resistance, whereas reintroduction of the gene in a CgSNQ2 deletion mutant fully reversed this effect. Expression of CgSNQ2 in a S. cerevisiae strain lacking PDR5 mediated not only resistance to azoles but also to 4-nitroquinoline N-oxide, which is a ScSNQ2-specific substrate. A putative gain-of-function mutation, P822L, was identified in CgPDR1 from BPY55. Disruption of CgPDR1 in BPY55 conferred enhanced azole susceptibility and eliminated CgSNQ2 expression, whereas introduction of the mutated allele in a susceptible strain where CgPDR1 had been disrupted conferred azole resistance and CgSNQ2 upregulation, indicating that CgSNQ2 was controlled by CgPDR1. Finally, CgSNQ2 was shown to be involved in the in vivo response to fluconazole. Together, our data first demonstrate that CgSNQ2 contributes to the development of CgPDR1-dependent azole resistance in C. glabrata. The overlapping in function and regulation between CgSNQ2 and ScSNQ2 further highlight the relationship between S. cerevisiae and C. glabrata.

SummaryResistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococcal meningitis, the most frequently encountered lifethreatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans , a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 ( CnAFR1 ). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans . Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene AFR1 in Cryptococcus neoformans is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of AFR1 in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of C. neoformans, strain BPY22; an afr1 mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an AFR1-overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22-and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the AFR1 gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of C. neoformans.Fluconazole (FLC) and other azole antifungal drugs are the agents most widely used for prevention and treatment of infections with Cryptococcus neoformans. Their confirmed efficacy and safety combined with their excellent pharmacokinetic profiles make them extremely important therapeutic options for the management of cryptococcosis at various body sites (6). In spite of their extensive use, resistance to these drugs among C. neoformans strains is apparently uncommon, although it has been implicated in several cases of treatment failure or infection relapse (3-4, 6, 20). Some authors have suggested that the frequency of resistance may have been underestimated because azole-resistant mutants of C. neoformans are often less virulent than their wild-type counterparts (6, 22). However, findings with other pathogenic yeast species demonstrate that antifungal resistance is not necessarily associated with attenuated pathogenicity (2, 44). For example, in a study of Candida albicans, Becker et al. (2) demonstrated that virulence is reduced by the disruption of the efflux pump-encoding MDR1 gene, whose overexpression leads to fluconazole resistance (36, 42). Graybill et al. (21) used a mouse model of systemic candidiasis to assess the virulences of a series of C. albicans isolates with increasing f...

Methionine sulfoxide reductases A and B are antioxidant repair enzymes that reduce the S-and R-diastereomers of methionine sulfoxides back to methionine, respectively. Enterococcus faecalis, an important nosocomial pathogen, has one msrA gene and one msrB gene situated in different parts of the chromosome. Promoters have been mapped and mutants have been constructed in two E. faecalis strains (strains JH2-2 and V583) and characterized. For both backgrounds, the mutants are more sensitive than the wild-type parents to exposure to H 2 O 2 , and in combination the mutations seem to be additive. The virulence of the mutants has been analyzed in four different models. Survival of the mutants inside mouse peritoneal macrophages stimulated with recombinant gamma interferon plus lipopolysaccharide but not in naïve phagocytes is significantly affected. The msrA mutant is attenuated in the Galleria mellonella insect model. Deficiency in either Msr enzyme reduced the level of virulence in a systemic and urinary tract infection model. Virulence was reconstituted in the complemented strains. The combined results show that Msr repair enzymes are important for the oxidative stress response, macrophage survival, and persistent infection with E. faecalis.

A low count of CD4 + and CD8 + lymphocytes is a hallmark laboratory finding in the coronavirus disease 2019 (COVID-19). Using flow cytometry, we observed significantly higher CD95 (Fas) and PD-1 expression on both CD4 + T and CD8 + T cells in 42 COVID-19 patients when compared to controls. Higher CD95 expression in CD4 + cells correlated with lower CD4 + counts. A higher expression of CD95 in CD4 + and CD8 + lymphocytes correlated with a lower percentage of naive events. Our results might suggest a shift to antigen-activated T cells, expressing molecules increasing their propensity to apoptosis and exhaustion during COVID-19 infection.

The aims of the study were to analyze the clinical and epidemiological characteristics and treatments for patients who developed zygomycosis enrolled in Italy during the European Confederation of Medical Mycology of medical mycology survey. This prospective multicenter study was performed between 2004 and 2007 at 49 italian Departments. 60 cases of zygomycosis were enrolled: the median age was 59.5 years (range 1-87), with a prevalence of males (70%). The majority of cases were immunocompromised patients (42 cases, 70%), mainly hematological malignancies (37). Among non-immunocompromised (18 cases, 30%), the main category was represented by patients with penetrating trauma (7/18, 39%). The most common sites of infection were sinus (35%) with/without CNS involvement, lung alone (25%), skin (20%), but in 11 cases (18%) dissemination was observed. According to EORTC criteria, the diagnosis of zygomycosis was proven in 46 patients (77%) and in most of them it was made in vivo (40/46 patients, 87%); in the remaining 14 cases (23%) the diagnosis was probable. 51 patients received antifungal therapy and in 30 of them surgical debridement was also performed. The most commonly used antifungal drug was liposomal amphotericin B (L-AmB), administered in 44 patients: 36 of these patients (82%) responded to therapy. Altogether an attributable mortality rate of 32% (19/60) was registered, which was reduced to 18% in patients treated with L-AmB (8/44). Zygomycosis is a rare and aggressive filamentous fungal infection, still associated with a high mortality rate. This study indicates an inversion of this trend, with a better prognosis and significantly lower mortality than that reported in the literature. It is possible that new extensive, aggressive diagnostic and therapeutic procedures, such as the use of L-AmB and surgery, have improved the prognosis of these patients.