Bloodstream infections (BSI) caused by extended-spectrum β-lactamase (ESBL)-producing organisms markedly increase the rates of treatment failure and death. We conducted a retrospective cohort analysis to identify risk factors for mortality in adult in-patients with BSI caused by ESBL-producing Enterobacteriaceae (ESBL-BSI). Particular attention was focused on defining the impact on the mortality of inadequate initial antimicrobial therapy (defined as the initiation of treatment with active antimicrobial agents >72 h after collection of the first positive blood culture). A total of 186 patients with ESBL-BSI caused by Escherichia coli ( n = 104), Klebsiella pneumoniae ( n = 58), or Proteus mirabilis ( n = 24) were identified by our microbiology laboratory from 1 January 1999 through 31 December 2004. The overall 21-day mortality rate was 38.2% (71 of 186). In multivariate analysis, significant predictors of mortality were inadequate initial antimicrobial therapy (odds ratio [OR] = 6.28; 95% confidence interval [CI] = 3.18 to 12.42; P < 0.001) and unidentified primary infection site (OR = 2.69; 95% CI = 1.38 to 5.27; P = 0.004). The inadequately treated patients (89 of 186 [47.8%]) had a threefold increase in mortality compared to the adequately treated group (59.5% versus 18.5%; OR = 2.38; 95% CI = 1.76 to 3.22; P < 0.001). The regimens most commonly classified as inadequate were based on oxyimino cephalosporin or fluoroquinolone therapy. Prompt initiation of effective antimicrobial treatment is essential in patients with ESBL-BSI, and empirical decisions must be based on a sound knowledge of the local distribution of pathogens and their susceptibility patterns.

Nosocomial Candida bloodstream infections rank among infections with highest mortality rates. A retrospective cohort analysis was conducted at Catholic University Hospital to estimate the risk factors for mortality of patients with candidemia. We reviewed records for patients with a Candida bloodstream infection over a 5-year period (January 2000 through December 2004). Two hundred ninety-four patients (42.1% male; mean age ± standard deviation, 65 ± 12 years) were studied. Patients most commonly were admitted with a surgical diagnosis (162 patients [55.1%]), had a central venous catheter (213 [72.4%]), cancer (118 [40.1%]), or diabetes (58 [19.7%]). One hundred fifty-four (52.3%) patients died within 30 days. Of 294 patients, 168 (57.1%) were infected by Candida albicans , 64 (21.7%) by Candida parapsilosis , 28 (9.5%) by Candida tropicalis , and 26 (8.8%) by Candida glabrata . When fungal isolates were tested for biofilm formation capacity, biofilm production was most commonly observed for isolates of C. tropicalis (20 of 28 patients [71.4%]), followed by C. glabrata (6 of 26 [23.1%]), C. albicans (38 of 168 [22.6%]), and C. parapsilosis (14 of 64 [21.8%]). Multivariable analysis identified inadequate antifungal therapy (odds ratio [OR], 2.35; 95% confidence interval [95% CI], 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.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different “hot spots,” were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions. Candida glabrata is a yeast causing several diseases in humans and especially in immuno-compromised people. C. glabrata infections are treated with antifungal agents, however the use of some agents, for example azoles, is associated with the development of resistance. In this yeast species, azole resistance is mediated almost exclusively by ATP Binding Cassette (ABC) multidrug transporters. Their overexpression results in enhanced efflux of azoles and thus generates resistance. Regulation of ABC transporters is therefore of pivotal importance to understanding azole resistance. In C. glabrata, the expression of ABC transporters is mediated by a zinc finger transcription factor called CgPDR1. Gain of function (GOF) mutations in CgPDR1 result in high ABC transporter expression. In this study, we investigated the occurrence of GOF mutations in a large collection of azole-resistant isolates and found a high variety of mutations localized in three distinct domains of CgPDR1. We found that these mutations are not only associated with resistance but also enhanced virulence and fitness of C. glabrata in animal models. Our study provides for the first time evidence that mutations causing antifungal resistance can also provide a selective advantage under host conditions and thus highlights the need of carefully monitoring resistance in this pathogen.

The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene ( CgERG11 ), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata , of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2 , alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2 . Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11 , were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2 , were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1 -, CgCDR2 -, and CgSNQ2 -encoded efflux pumps might explain the azole resistance in our set of isolates.