Multiresistance to Nonazole Fungicides in Aspergillus fumigatus TR34/L98H Azole-Resistant Isolates.

Drug resistance is a worldwide problem affecting all pathogens. The human fungal pathogen Aspergillus fumigatus coexists in the environment with other fungi targeted by crop protection compounds, being unintentionally exposed to the selective pressure of multiple antifungal classes and leading to the selection of resistant strains. A. fumigatus azole-resistant isolates are emerging in both clinical and environmental settings. Since their approval, azole drugs have dominated clinical treatment for aspergillosis infections and the agriculture fungicide market. However, other antifungal classes are used for crop protection, including benzimidazoles (methyl benzimidazole carbamates [MBCs]), strobilurins (quinolone oxidation inhibitors [QoIs]), and succinate dehydrogenase inhibitors (SDHIs). Mutations responsible for resistance to these fungicides have been widely researched in plant pathogens, but resistance has not been explored in A. fumigatus. In this work, the genetic basis underlying resistance to MBCs, QoIs, and SDHIs was studied in azole-susceptible and -resistant A. fumigatus strains. E198A/Q and F200Y mutations in ß-tubulin conferred resistance to MBCs, G143A and F129L substitutions in cytochrome b conferred resistance to QoIs, and H270R/Y mutations in SdhB conferred resistance to SDHIs. Characterization of susceptibility to azoles showed a correlation between strains resistant to these fungicides and the ones with tandem-repeat (TR)-based azole resistance mechanisms. Whole-genome sequencing analysis showed a genetic relationship among fungicide multiresistant strains, which grouped into subclusters that included only strains carrying the TR-based azole resistance mechanisms, indicating a common ancestor/evolution pattern and confirming the environmental origin of this type of azole-resistant A. fumigatus.

Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus.

The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53 Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.

To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles.

Rapid development of Candida krusei echinocandin resistance during caspofungin therapy.

In invasive candidiasis, there has been an epidemiological shift from Candida albicans to non-albicans species infections, including infections with C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. Although the prevalence of C. krusei remains low among yeast infections, its intrinsic resistance to fluconazole raises epidemiological and therapeutic concerns. Echinocandins have in vitro activity against most Candida spp. and are the first-line agents in the treatment of candidemia. Although resistance to echinocandin drugs is still rare, individual cases of C. krusei resistance have been reported in recent years, especially with strains that have been under selective pressure. A total of 15 C. krusei strains, isolated from the blood, urine, and soft tissue of an acute lymphocytic leukemia patient, were analyzed. Strains developed echinocandin resistance during 10 days of caspofungin therapy. The molecular epidemiology of the isolates was investigated using two different typing methods: PCR-based amplification of the species-specific repetitive polymorphic CKRS-1 sequence and multilocus sequence typing. All isolates were genetically related, and the mechanism involved in decreased echinocandin susceptibility was characterized. Clinical resistance was associated with an increase in echinocandin MICs in vitro and was related to three different mutations in hot spot 1 of the target enzyme Fks1p. Molecular evidence of the rapid acquisition of resistance by different mutations in FKS1 highlights the need to monitor the development of resistance in C. krusei infections treated with echinocandin drugs.

An invertebrate model to evaluate virulence in Aspergillus fumigatus: the role of azole resistance.

The impact of different mutations in the Aspergillus fumigatus ergosterol biosynthesis pathway on pathogenesis has been evaluated using a simple invertebrate mini host, the caterpillar Galleria mellonella. A set of strains that includes clinical isolates and isogenic mutants with mutations at the cyp51A gene conferring azole resistance were studied. All strains demonstrated a similar in vitro growth pattern and are equally virulent against the insect larvae. These results suggest that in A. fumigatus acquisition of this particular azole-resistance mechanism would not imply any significant change in virulence. G. mellonella may provide a convenient and inexpensive model for the in vivo prescreening of mutants of A. fumigatus, contributing to the generation of a hypotheses that can be further tested in refined experiments in mammalian models.

Population-based survey of filamentous fungi and antifungal resistance in Spain (FILPOP Study).

A population-based survey was conducted to investigate the epidemiology of and antifungal resistance in Spanish clinical strains of filamentous fungi isolated from deep tissue samples, blood cultures, and respiratory samples. The study was conducted in two different periods (October 2010 and May 2011) to analyze seasonal variations. A total of 325 strains were isolated in 29 different hospitals. The average prevalence was 0.016/1,000 inhabitants [corrected]. Strains were identified by sequencing of DNA targets and susceptibility testing by the European Committee for Antimicrobial Susceptibility Testing reference procedure. The most frequently isolated genus was Aspergillus, accounting for 86.3% of the isolates, followed by Scedosporium at 4.7%; the order Mucorales at 2.5%; Penicillium at 2.2%, and Fusarium at 1.2%. The most frequent species was Aspergillus fumigatus (48.5%), followed by A. flavus (8.4%), A. terreus (8.1%), A. tubingensis (6.8%), and A. niger (6.5%). Cryptic/sibling Aspergillus species accounted for 12% of the cases. Resistance to amphotericin B was found in 10.8% of the isolates tested, while extended-spectrum triazole resistance ranged from 10 to 12.7%, depending on the azole tested. Antifungal resistance was more common among emerging species such as those of Scedosporium and Mucorales and also among cryptic species of Aspergillus, with 40% of these isolates showing resistance to all of the antifungal compounds tested. Cryptic Aspergillus species seem to be underestimated, and their correct classification could be clinically relevant. The performance of antifungal susceptibility testing of the strains implicated in deep infections and multicentric studies is recommended to evaluate the incidence of these cryptic species in other geographic areas.

Candida tropicalis antifungal cross-resistance is related to different azole target (Erg11p) modifications.

Candida tropicalis ranks between third and fourth among Candida species most commonly isolated from clinical specimens. Invasive candidiasis and candidemia are treated with amphotericin B or echinocandins as first-line therapy, with extended-spectrum triazoles as acceptable alternatives. Candida tropicalis is usually susceptible to all antifungal agents, although several azole drug-resistant clinical isolates are being reported. However, C. tropicalis resistant to amphotericin B is uncommon, and only a few strains have reliably demonstrated a high level of resistance to this agent. The resistance mechanisms operating in C. tropicalis strains isolated from clinical samples showing resistance to azole drugs alone or with amphotericin B cross-resistance were elucidated. Antifungal drug resistance was related to mutations of the azole target (Erg11p) with or without alterations of the ergosterol biosynthesis pathway. The antifungal drug resistance shown in vitro correlated very well with the results obtained in vivo using the model host Galleria mellonella. Using this panel of strains, the G. mellonella model system was validated as a simple, nonmammalian minihost model that can be used to study in vitro-in vivo correlation of antifungals in C. tropicalis. The development in C. tropicalis of antifungal drug resistance with different mechanisms during antifungal treatment has potential clinical impact and deserves specific prospective studies.

Analysis and characterization of cultivable extremophilic hydrolytic bacterial community in heavy-metal-contaminated soils from the Atacama Desert and their biotechnological potentials.

AIMS: To isolate and characterize the cultivable community of hydrolase producers (amylase, protease, lipase, DNase, xylanase and pullulanase) inhabiting heavy-metal-contaminated soils in extreme conditions from the Atacama Desert. METHODS AND RESULTS: A total of 25 bacterial strains showing hydrolytic activities have been selected including halotolerants, extremely halotolerants and moderate halophiles. Most hydrolase producers were assigned to the family B acillaceae, belonging to the genera Bacillus (nine strains), Halobacillus (seven strains) and Thalassobacillus (five strains) and four isolates were related to members of the families Pseudomonadaceae, Halomonadaceae and Staphylococcaceae. The selected strains were then characterized for their tolerance pattern to six heavy metals, measured as minimal inhibitory concentrations (MICs). CONCLUSIONS: The diversity found in the cultivable bacterial community analysed is more limited than that detected in other ecological studies owing to the restrictive conditions used in the screening. The dominant bacteria were Firmicutes and particularly, species related to the genus Bacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is focused on the characterization of extremophilic hydrolytic bacteria, providing candidates as a source of novel enzymes with biotechnological applications.

Zygomycosis caused by Rhizopus microsporus and Rhizopus oryzae in Madhya Pradesh (M.P.) Central India: a report of two cases.

Zygomycosis encompasses infections due to two distinct orders of fungi, Mucorales and Entomophthorales. With rare exception, Entomophthorales are restricted to tropical areas. By contrast, mucorales are ubiquitous opportunistic fungi, which play a crucial part in the natural decay process. In human pathology, they may be opportunistic agents and be responsible for rare infection called (Mucormycosis) zygomycosis. We report two cases of zygomycosis from Madhya Pradesh, Central India, one caused by Rhizopus oryzae in a diabetic patient and another caused by Rhizopus microsporus in an apparently healthy patient. The cases were diagnosed by direct microscopy, histopathological examination and culture. Both the patients were successfully treated with liposomal amphotericin B. Rhizopus microsporus is, for the first time reported from Madhya Pradesh, India, causing rhino-maxillary orbital zygomycosis.

Frequency of voriconazole resistance in vitro among Spanish clinical isolates of Candida spp. According to breakpoints established by the Antifungal Subcommittee of the European Committee on Antimicrobial Susceptibility Testing.

A total of 4,226 Spanish clinical isolates of Candida spp. were analyzed to assess resistance to voriconazole according to breakpoints established by the European Committee for Antimicrobial Susceptibility Testing (where susceptibility [S] to voriconazole corresponds to a MIC of ≤ 0.12 mg/liter). Resistance was uncommon among Candida albicans (5%), C. parapsilosis (1.2%), and C. tropicalis (11%) isolates. Voriconazole MICs of >0.12 mg/liter were more frequent among Candida glabrata and C. krusei isolates. A significant percentage of voriconazole-resistant strains came from oropharyngeal infections and exhibited high MICs of other azoles.

Probing the role of point mutations in the cyp51A gene from Aspergillus fumigatus in the model yeast Saccharomyces cerevisiae.

Azole-resistant strains of Aspergillus fumigatus have been detected and the underlying molecular mechanisms of resistance characterized. Point mutations in the cyp51A gene have been proved to be related to azole resistance in A. fumigatus clinical strains and with different resistance profiles depending on the amino acid change (G54E, G54V, G54R, G54W, M220V, M220K, M220T, M220I). The aim of this work was to express A. fumigatus cyp51A genes in the yeast Saccharomyces cerevisiae in order to better assess the contribution of each independent amino acid substitution to resistance. A tetracycline regulatable system allowing repression of the endogenous essential ERG11 gene was used. The expression of Aspergillus cyp51A alleles could efficiently restore the absence of ERG11 in S. cerevisiae. In general, S. cerevisiae clones expressing. A. fumigatus cyp51A alleles from azole-resistant isolates showed higher MICs to all azoles tested than those expressing alleles from susceptible isolates. The azole susceptibility profiles obtained in S. cerevisiae upon expression of specific cyp51A alleles recapitulated susceptibility profiles observed from their A. fumigatus origins. In conclusion this work supports the concept that characteristics of specific A. fumigatus cyp51A alleles could be investigated in the heterologous host S. cerevisiae.

TREM1 regulates antifungal immune responses in invasive pulmonary aspergillosis.

Pattern recognition receptors (PRRs) are responsible for Aspergillus fumigatus recognition by innate immunity and its subsequent immune signaling. The triggering receptor expressed on myeloid cells 1 (TREM1) is a recently characterized pro-inflammatory receptor constitutively expressed on the surface of neutrophils and macrophages. A soluble form (sTREM1) of this protein that can be detected in human body fluids has been identified. Here we investigated the role of TREM1 during invasive pulmonary aspergillosis (IPA). IPA patients displayed significantly higher levels of sTREM1 in bronchoalveolar lavages when compared to control patients. Functional analysis in TREM1 showed that the levels of sTREM1 and TREM1 pathway-related cytokines were influenced by single nucleotide polymorphisms in TREM1. In addition, we confirmed a role of TREM1 on antifungal host defense against A. fumigatus in a murine model of IPA. TREM1 deficiency increased susceptibility to infection in the immunosuppressed murine host. Deletion of TREM1 showed delayed innate and adaptive immune responses and impaired pro-inflammatory cytokine responses. The absence of TREM1 in primary macrophages attenuated the TLR signaling by altering the expression of both receptor and effector proteins that are critical to the response against A. fumigatus. In this study, and for the first time, we demonstrate the key role for the TREM1 receptor pathway during IPA.

Interlaboratory reproducibility of a single-locus sequence-based method for strain typing of Aspergillus fumigatus.

Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA_3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

Blood and tissue distribution of posaconazole in a rat model of invasive pulmonary aspergillosis.

Posaconazole is the recommended prophylactic agent in patients at high risk of invasive fungal infection, since adequate drug levels seem to be reached in target sites despite the relatively low levels detected in blood. The objective of this study is to obtain pharmacokinetic (PK) information associated to blood and tissue distribution of posaconazole in an animal model of invasive pulmonary aspergillosis. The PK parameters in lung samples were systematically higher than in serum. After multiple-dose administration of posaconazole, a significant accumulation of the drug was evident in lung tissue. The PK behavior of posaconazole in this particular experimental model is similar to that observed in humans. Thus, we believe this model could be a valid tool to evaluate posaconazole exposure-response relationship.

New resistance mechanisms to azole drugs in Aspergillus fumigatus and emergence of antifungal drugs-resistant A. fumigatus atypical strains.

Azole drug resistance in Aspergillus fumigatus is an uncommon but well-known phenomenon. The analysis of resistance mechanisms at molecular level has identified the bases for A. fumigatus azole resistance. To date, the most prevalent mechanism of azole resistance appears to be the modification of Cyp51, specifically mutations in cyp51A gene. These mutations have been associated with three different antifungal susceptibility profiles: (i) cross-resistance to itraconazole and posaconazole that has been associated with amino acid substitutions at glycine 54 (G54), (ii) elevated MICs to all azole drugs associated with amino acid substitutions at methionine M220, and (iii) cross-resistance to all azole drugs related to the presence of Cyp51A substitutions at leucine 98 for histidine (L98H) linked to a duplication in tandem of a 34 bp repeat in the cyp51A promoter region, which seem to be responsible for increased cyp51A gene expression. Another matter of concern is the increasing reports of isolation of genetic variants of A. fumigatus, originally misidentified as poorly sporulating strains of A. fumigauts, as a causative agents of invasive infection. Many of these isolates belonging to the Aspergillus section Fumigati have been found to be resistant in vitro to multiple antifungal drugs. Current data show that susceptibility profile of these variants could be predictable depending on the species. Resistance among clinical strains of filamentous fungi may become more common in the future associated with the spread of prophylaxis, pre-emptive treatments and specific therapies with antifungal agents.

Corrigendum to "First detection of Aspergillus fumigatus azole resistant strain due to Cyp51A TR46/Y121F/T289A in an azole-naive patient in Spain" [New Microbes New Infect 6 (2015) 33-34].

[This corrects the article DOI: 10.1016/j.nmni.2015.04.005.].

Correlation between the procedure for antifungal susceptibility testing for Candida spp. of the European Committee on Antibiotic Susceptibility Testing (EUCAST) and four commercial techniques.

The correlation between results obtained with the European Committee on Antibiotic Susceptibility Testing (EUCAST) antifungal susceptibility testing procedure (document 7.1) and four commercial systems was evaluated for a collection of 93 clinical isolates of Candida spp. Overall, agreement between the EUCAST procedure and the Sensititre YeastOne and Etest methods was 75% and 90.4%, respectively. The correlation indices (p < 0.01) between the EUCAST and commercial methods were 0.92 for Sensititre YeastOne, 0.89 for Etest, - 0.90 for Neo-Sensitabs, and 0.95 for Fungitest. Amphotericin B MICs obtained by Sensititre YeastOne were consistently higher than with the EUCAST method and, although very major errors were not observed, 91% of MICs were misclassified. Amphotericin B- and fluconazole-resistant isolates were identified correctly with Sensititre YeastOne, Etest and Fungitest. Neo-Sensitabs identified amphotericin B-resistant isolates, but misclassified > 5% of fluconazole-resistant isolates as susceptible. The commercial methods, particularly Etest and Fungitest, appeared to be suitable alternatives to the EUCAST procedure for antifungal susceptibility testing of clinical isolates of Candida.

An alternative host model of a mixed fungal infection by azole susceptible and resistant Aspergillus spp strains.

Aspergillus fumigatus is the most common mold involved in human infections. However, the number of non-fumigatus species able to cause disease is continuously increasing. Among them, Aspergillus lentulus is reported in hematological and cystic fibrosis patients and in those treated with corticosteroids. A. lentulus differs from A. fumigatus in some clinically relevant aspects such as virulence and antifungal susceptibility, showing high MICs to most antifungals. Previous studies proved that A. lentulus was pathogenic in immunocompromised mice, although the course of the infection was delayed compared to A. fumigatus. These differences could explain why A. lentulus is mostly found in mixed infections with A. fumigatus challenging the diagnosis and treatment. We used the alternative model host Galleria mellonella to compare virulence, host interaction, fungal burden and antifungal response when larvae were infected with A. fumigatus or A. lentulus alone, and with a mixture of both species. A. lentulus was pathogenic in G. mellonella but infected larvae did not respond to therapeutic doses of voriconazole. We were able to simultaneously detect A. fumigatus and A. lentulus by a multiplex Nested Real Time PCR (MN-PCR). Comparative analysis of larvae histological sections showed melanization of both species but presented a different pattern of immune response by haemocytes. Analysis of fungal burden and histology showed that A. lentulus survived in the G. mellonella despite the antifungal treatment in single and mixed infections. We conclude that the simultaneous presence of antifungal susceptible and resistant Aspergillus species would likely complicate the management of these infections.

In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection.

The emergence of azole resistance in Aspergillus fumigatus is a clinically important issue in the management of invasive aspergillosis as it could limit therapeutic options. Accurate measurement of in vitro antifungal activity in terms of minimum inhibitory concentration (MIC) is considered of clinical relevance and often gives useful therapeutic information for physicians. However, the lack of in vitro-in vivo correlation is frequent and the observed in vitro phenotype does not always correlate with the in vivo response. In this regard, a wild-type strain and five A. fumigatus cyp51A mutated strains showing different azole susceptibility profiles were used to investigate whether the greater wax moth (Galleria mellonella) is an alternative model to assess the in vivo efficacy of voriconazole and posaconazole. Administration of both azoles improved the survival of larvae infected with susceptible strains. However, those larvae infected with resistant strains did not respond to treatment. The phenotype observed in vitro was found to correlate with the efficacy observed in vivo. Moreover, using this in vivo model, the pharmacodynamic target predicting therapeutic success (AUC(0-24)/MIC) was in the same range as previously described, allowing the use of the G. mellonella model to predict the azole susceptibility profile of A. fumigatus strains.