Azole-resistant Aspergillus fumigatus in the Italian environment.

OBJECTIVES: Azole resistance in Aspergillus fumigatus environmental and clinical isolates is recognised as an emerging problem worldwide. Development of azole resistance may be environmentally driven because of the massive use of azole fungicides in agriculture. The mechanism of azole resistance is mostly related to mutations in the cyp51A gene. METHODS: A. fumigatus azole resistance in the environment was previously documented in northern Italy. This study extended the research in the agricultural environment also in central and southern Italy and investigated differences in the Italian geographical areas and in the different types of crops. RESULTS: A total of 177 samples (173 soil samples and 4 Dutch bulbs) collected in the period 2014-20117 in 14 Italian regions were analysed. Itraconazole-resistant A. fumigatus isolates grew in 16.9% of the screened samples. Differences were observed in soil samples from the three Italian geographic areas: 12.5% in the north, 15.2% in the centre and 24.1% in the south. Resistant isolates were from different cultivations, treated or officially not treated with azole fungicides. Sequencing of the cyp51A gene confirmed that resistance was mainly associated with the TR34/L98H mutation (29/30 isolates); 1 isolate showed the G54E mutation. CONCLUSIONS: The risk for patients to acquire multi-azole-resistant strains from the environment could have a serious impact on the management of life-threatening invasive infections. The azole resistance rate of 16.9% found in Italy requires suitable monitoring of antifungal susceptibility of clinical isolates.

Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples.

The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose.

Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon?

Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results: The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions:Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole.

Antibiotic resistance: Italian awareness survey 2016.

Antimicrobial resistance has become a public health priority worldwide. The WHO conducted a survey concerning the personal use of antibiotics, knowledge of appropriate use and awareness of the issue of resistance. A similar survey was conducted in Italy involving 666 young university students and 131 seniors attending courses of the University of the third age. Antibiotics seem to be taken with moderate frequency and appropriately: 30% of respondents took them in the past six months and 94% took these drugs only prescribed by a doctor, in the correct dose and for the proper duration. Notable confusion concerning the conditions treatable with antibiotics was detected (only 30% indicated gonorrhea, and 30-40% believed that antibiotics should be employed for fever, cold, and flu), while 94% of participants seemed aware of the problem of antibiotic resistance. Most of the respondents identified the behaviors that can reduce the phenomenon of resistance (regular handwashing and use of antibiotics only when prescribed and needed). The results of our survey, that involved people of high level of instruction and living in urban areas of northern regions, cannot be extended to all the Italian population. However, they provide valid elements to promote initiatives aimed to a more aware use of antibiotics.

Corrigendum: Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon?

[This corrects the article DOI: 10.3389/fmicb.2018.00516.].