Summary The epidemiology of Candida infections has changed in recent years. Although Candida albicans is still the main cause of invasive candidiasis in most clinical settings, a substantial proportion of patients is now infected with non‐albicans Candida species. The various Candida species vary in their susceptibility to the most commonly used antifungal agents, and the intrinsic resistance to antifungal therapy seen in some species, along with the development of acquired resistance during treatment in others, is becoming a major problem in the management of Candida infection. A better understanding of the mechanisms and clinical impact of antifungal drug resistance is essential for the efficient treatment of patients with Candida infection and for improving treatment outcomes. Herein, we report resistance to the azoles and echinocandins among Candida species.

A total of 216 patients (mean age 63.4 ± 18.5 years; 58.3 % males) were included in the study. Of these, 163 (75 %) were admitted to the intensive care unit. Overall 30-day mortality was 54 %. Significantly higher Acute Physiology and Chronic Health Evaluation (APACHE) II scores, dysfunctional organs, and inadequate antifungal therapy were compared in nonsurvivors and survivors. No differences in survivors versus nonsurvivors were found in terms of the time from positive blood culture to initiation of adequate antifungal therapy. Multivariate logistic regression identified inadequate source control, inadequate antifungal therapy, and 1-point increments in the APACHE II score as independent variables associated with a higher 30-day mortality rate.

Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis.Enterococcus faecalis is a natural member of the intestinal microflora of warm-blooded animals and humans. However, E. faecalis remains an important opportunistic pathogen and represents one of the principal causes of nosocomial infections in the United States and Europe (33; for a review, see reference 20). Especially for immunocompromised patients, these infections include endocarditis, meningitis, pneumonia, peritonitis, visceral abscesses, urinary infections, and septicemia (13). About a dozen putative virulence factors have been identified in E. faecalis, but mechanisms of virulence remain not fully understood. These factors are involved in different steps of the infection process, such as attachment to host cells or extracellular matrix (ECM), macrophage resistance, tissue damage, and immune system evasion (20).For extracellular pathogens such as E. faecalis or Staphylococcus aureus, components of ECM or serum (i.e., collagen, fibronectin, and fibrinogen) are preferred targets for adhesion. During important steps of the infectious process, they interact with bacterial surface-exposed molecules, which include microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Ace, an adhesin that binds collagen (types I and IV) and laminin and belongs to the MSCRAMM family, was identified in E. faecalis by sequence homology with the virulence factor Cna, a well-characterized MSCRAMM in S. aureus (16,26). Synthesis of the A domain of Ace by S. aureus increased its arthritogenic potential to a level similar to that of S. aureus expressing Cna (34). Study of the sequence diversity of ace among several strains of E. faecalis has revealed an important variation in the number of repeated sequences (17). However, the role played by these regions has not yet been elucidated. The expression of Ace is significantly induced by high temperature (culture at 46°C) and in vivo by the presence of serum or ECM com...

eThe widespread use of antifungal agents, which is likely to expand with their enhanced availability, has promoted the emergence of drug-resistant strains. Antifungal susceptibility testing (AFST) is now an essential procedure for guiding appropriate antifungal therapy. Recently, we developed a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method that enables the detection of fungal isolates with reduced echinocandin susceptibility, relying on the proteome changes that are detectable after a 15-h exposure of fungal cells to serial drug concentrations. Here, we describe a simplified version of this approach that facilitates discrimination of the susceptible and resistant isolates of Candida albicans after a 3-h incubation in the presence of "breakpoint" level drug concentrations of the echinocandin caspofungin (CSF). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) g/ml of CSF were used to create individual composite correlation index (CCI) matrices for 65 C. albicans isolates, including 13 fks1 mutants. Isolates are then classified as susceptible or resistant to CSF if the CCI values of spectra at 0.03 and 32 g/ml are higher or lower, respectively, than the CCI values of spectra at 0.03 and 0 g/ml. In this way, the drug resistance of C. albicans isolates to echinocandin antifungals can be quickly assessed. Furthermore, the isolate categorizations determined using MALDI-TOF MS-based AFST (ms-AFST) were consistent with the wild-type and mutant FKS1 genotypes and the AFST reference methodology. The ms-AFST approach may provide a rapid and reliable means of detecting emerging antifungal resistance and accelerating the initiation of appropriate antifungal treatment.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility.

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...

In order to identify regulators of the oxidative stress response in Enterococcus faecalis, an important human pathogen, several genes annotated as coding for transcriptional regulators were inactivated by insertional mutagenesis. One mutant, affected in the ef2958 locus (designated hypR [hydrogen peroxide regulator]), appeared to be highly sensitive to oxidative challenge caused by hydrogen peroxide. Moreover, testing of the hypR mutant by using an in vivo-in vitro macrophage infection model resulted in a highly significant reduction in survival compared to the survival of parent strain JH2-2. Northern blot analyses were carried out with probes specific for genes encoding known antioxidant enzymes, and they showed that the ahpCF (alkyl hydroperoxide reductase) transcript was expressed less in mutant cells. Mobility shift protein-DNA binding assays revealed that HypR regulated directly the expression of hypR itself and the ahpCF operon. Our combined results showed that HypR appeared to be directly involved in the expression of ahpCF genes under oxidative stress conditions and suggested that this regulator could contribute to the virulence of E. faecalis.

Since its first description, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide with over 2 million cases reported and thousands of deaths. Every human is susceptible to the infection, and pregnant women are not spared. Although reports documented COVID-19 infections among pregnant women 1,2 and described the neonatal outcome relatively to early days of life, 3 to date, no information on late-onset infection in newborns to mother with SARS-CoV-2 contracted in pregnancy are available. AbstractObjective To date, no information on late-onset infection in newborns to mother with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contracted in pregnancy are available. This study aimed to evaluate postdischarge SARS-CoV-2 status of newborns to mothers with COVID-19 in pregnancy that, at birth, were negative to SARS-CoV-2. Study Design This is an observational study of neonates born to mothers with coronavirus disease 2019 .Results Seven pregnant women with documented SARS-CoV-2 infection have been evaluated in our institution. One woman had a spontaneous abortion at 8 weeks of gestational age, four women recovered and are still in follow-up, and two women delivered. Two newborns were enrolled in the study. At birth and 3 days of life, newborns were negative to SARS-CoV-2. At 2-week follow-up, one newborn tested positive although asymptomatic. Conclusion Our findings highlight the importance of follow-up of newborns to mothers with COVID-19 in pregnancy, since they remain at risk of contracting the infection in the early period of life and long-term consequences are still unknown.