Evaluation of Synergistic Activity of Isavuconazole or Voriconazole plus Anidulafungin and the Occurrence and Genetic Characterization of Candida auris Detected in a Surveillance Program.

A total of 15 Candida auris isolates from the SENTRY antimicrobial surveillance program between 2006 and 2019 were combined with 21 isolates from other collections for the evaluation of antifungal susceptibility and synergy against anidulafungin plus voriconazole or isavuconazole using the checkerboard method. Surveillance isolates were analyzed for genetic relatedness and resistance mechanisms. Applying the tentative statistical epidemiological cutoff values and the Centers for Disease Control tentative breakpoints, 32/36 isolates were resistant to fluconazole, 5/36 were resistant to amphotericin B, 5/36 were non-wild-type (NWT) to anidulafungin, 3/36 were NWT to micafungin, and 1/36 and 10/36 were NWT to isavuconazole and voriconazole, respectively. Of these, 10 isolates were multidrug resistant, which means that these isolates were resistant to 2 antifungal classes. Synergy or partial synergy was noted in 5/36 and 22/36, respectively, of the isolates with the combination of anidulafungin plus voriconazole, and 11/36 and 19/36 isolates, respectively, for the combination of anidulafungin plus isavuconazole. Multilocus sequence type (MLST) analysis of the 15 SENTRY isolates demonstrated that the isolates from the US were genetically related to, but different from, isolates from Latin America (Panama and Colombia) and Germany. Single nucleotide polymorphism (SNP) analysis showed that the 15 SENTRY isolates belonged to the described international clades and had associated Erg11 alterations, including 11 isolates displaying K143R, one displaying F126L, and one displaying Y501H alterations and a fluconazole MIC result of ≥64 mg/liter. Resistance mechanisms were not observed in the two isolates displaying fluconazole MIC values at 4 and 16 mg/liter. Isavuconazole displayed activity and greater synergy when tested with anidulafungin than seen with anidulafungin plus voriconazole against the C. auris clinical isolates that displayed resistance phenotypes.

Isavuconazole nonwildtype Aspergillus fumigatus isolates from a global surveillance study display alterations in multiple genes involved in the ergosterol biosynthesis pathway not previously associated with resistance to other azoles.

OBJECTIVES: We evaluated 35 azole nonwildtype Aspergillus fumigatus isolates that were collected during 2017-2018 using whole genome sequencing (WGS) to detect alterations in the genes involved in the ergosterol biosynthesis pathway as well as other genes associated with azole resistance. METHODS: Among 297 A fumigatus isolates collected worldwide, 36 isolates displayed nonwildtype MIC values to isavuconazole, itraconazole, or voriconazole when tested by the CLSI reference broth microdilution method. Isolates were submitted to WGS and results were compared to 2 azolewildtype isolates. RESULTS: Among the 35 sequenced isolates (1 failed to produce quality sequences), 29 were nonwildtype to isavuconazole, 16 were nonwildtype to itraconazole, and 9 were nonwildtype to voriconazole (CLSI M59Ed2 criteria). A total of 9 isolates carried Cyp51A TR34/L98H alterations (8 from Italy and 1 from Belgium) and had nonwildtype MIC values for ≥2 azoles. A Cyp51B Q42L mutation was detected in 3 isolates, 1 nonwildtype voriconazole and 2 nonwildtype isavuconazole isolates. Three isolates harboured multiple mutations in Cyp51A (F46Y, M172V, E427K ± N248T, and D255E), including 1 isolate with the Cyp51B Q42L mutation. Mutations causing frameshifts, early termination, and duplications were observed among several genes and were more prevalent in isavuconazole nonwildtype isolates (66.7%) than in the isolates that were nonwildtype to 1 or 2 other azoles (22.2%). Nine isolates harboured frameshift mutations in a ERG25 homologue that is usually associated with changes in other genes and should be further evaluated. CONCLUSIONS: Cyp51A L98H/TR34 was the most common alteration observed among the azole nonwildtype A fumigatus isolates from a large surveillance study; however, only isolates that were nonwildtype to isavuconazole had alterations in multiple analysed genes. These isolates deserve further evaluation.

Activity of plazomicin compared with other aminoglycosides against isolates from European and adjacent countries, including Enterobacteriaceae molecularly characterized for aminoglycoside-modifying enzymes and other resistance mechanisms.

Background: Plazomicin is a next-generation aminoglycoside that was developed to overcome common aminoglycoside-resistance mechanisms. Objectives: We evaluated the activity of plazomicin and comparators against clinical isolates collected from 26 European and adjacent countries during 2014 and 2015 as part of the Antimicrobial Longitudinal Evaluation and Resistance Trends (ALERT) global surveillance programme. Methods: All 4680 isolates collected from 45 hospitals were tested for susceptibility to antimicrobials using the reference broth microdilution method. Selected isolates were screened for genes encoding carbapenemases, aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases. Results: Plazomicin (MIC50/90 0.5/2 mg/L) inhibited 95.8% of Enterobacteriaceae at ≤2 mg/L, including carbapenem-resistant Enterobacteriaceae (MIC50/90 0.25/128 mg/L). Plazomicin was more active compared with other aminoglycosides against isolates carrying blaKPC (MIC50/90 0.25/2 mg/L), isolates carrying blaOXA-48-like (MIC50/90 0.25/16 mg/L) and carbapenemase-negative isolates (MIC50/90 0.25/1 mg/L). Approximately 60% of the isolates harbouring blaVIM and blaNDM-1 carried 16S rRNA methyltransferases (mainly rmtB and armA). AME genes were detected among 728 isolates and 99.0% of these were inhibited by plazomicin at ≤2 mg/L. Plazomicin activity against Pseudomonas aeruginosa (MIC50/90 4/8 mg/L) was similar to amikacin activity (MIC50/90 2/16 mg/L). Plazomicin demonstrated activity against CoNS (MIC50/90 0.12/0.25 mg/L) and Staphylococcus aureus (MIC50/90 0.5/1 mg/L). Plazomicin activity was limited against Acinetobacter spp. (MIC50/90 8/>128 mg/L), Enterococcus spp. (MIC50/90 32/128 mg/L) and Streptococcus pneumoniae (MIC50/90 32/64 mg/L). Conclusions: Plazomicin demonstrated activity against Enterobacteriaceae isolates tested in this study, including isolates carrying AMEs and a high percentage of the carbapenem-non-susceptible isolates. Plazomicin displayed activity against staphylococci.

Monitoring Antifungal Resistance in a Global Collection of Invasive Yeasts and Molds: Application of CLSI Epidemiological Cutoff Values and Whole-Genome Sequencing Analysis for Detection of Azole Resistance in Candida albicans.

The activity of 7 antifungal agents against 3,557 invasive yeasts and molds collected in 29 countries worldwide in 2014 and 2015 was evaluated. Epidemiological cutoff values (ECVs) published in the Clinical and Laboratory Standards Institute (CLSI) M59 document were applied for species with no clinical breakpoints. Echinocandin susceptibility rates were 95.9% to 100.0% for the 5 most common Candida species, except for the rates for Candida parapsilosis to anidulafungin (88.7% susceptible, 100.0% wild type). Rates of fluconazole resistance ranged from 8.0% for Candida glabrata to 0.4% for Candida albicans Seven Candida species displayed 100.0% wild-type amphotericin B MIC results, and Candida dubliniensis and Candida lusitaniae exhibited wild-type echinocandin MIC results. The highest fluconazole, voriconazole, and posaconazole MIC values for Cryptococcus neoformans var. grubii were 8 µg/ml, 0.12 µg/ml, and 0.25 µg/ml, respectively. Aspergillus fumigatus isolates were 100.0% wild type for caspofungin and amphotericin B, but 3 (0.8%) of these isolates were non-wild type to itraconazole (2 isolates) or voriconazole (1 isolate). Mutations in FKS hot spot (HS) regions were detected among 13/20 Candida isolates displaying echinocandin MICs greater than the ECV (16 of these 20 isolates were C. glabrata). Most isolates carrying mutations in FKS HS regions were resistant to 2 or more echinocandins. Five fluconazole-nonsusceptible C. albicans isolates were submitted to whole-genome sequencing analysis. Gain-of-function, Erg11 heterozygous, and Erg3 homozygous mutations were observed in 1 isolate each. One isolate displayed MDR1 promoter allele alterations associated with azole resistance. Elevated levels of expression of MDR1 or CDR2 were observed in 3 isolates and 1 isolate, respectively. Echinocandin and azole resistance is still uncommon among contemporary fungal isolates; however, mechanisms of resistance to antifungals were observed among Candida spp., showing that resistance can emerge and monitoring is warranted.

High Rates of Nonsusceptibility to Ceftazidime-avibactam and Identification of New Delhi Metallo-ß-lactamase Production in Enterobacteriaceae Bloodstream Infections at a Major Cancer Center.

Resistance to the novel ß-lactam/ß-lactamase inhibitor combination ceftazidime-avibactam (CAZ-AVI) among carbapenem-resistant Enterobacteriaceae (CRE) has infrequently been reported in the United States. We report unexpectedly high rates of resistance to CAZ-AVI in CRE bloodstream isolates at our institution associated with the nonoutbreak spread of New Delhi metallo-ß-lactamase in diverse Enterobacteriaceae species.

Klebsiella pneumoniae Isolate from a New York City Hospital Belonging to Sequence Type 258 and Carrying blaKPC-2 and blaVIM-4.

Among 69 of 139 (49.6%) carbapenem-nonsusceptible Enterobacteriaceae carrying blaKPC, 1 Klebsiella pneumoniae was also positive for blaVIM. The isolate belonged to sequence type 258 (ST258) and carried blaKPC-2 on a copy of Tn4401a and blaVIM-4 on a class 1 integron. Genes were located on distinct plasmids belonging to Inc types A/C and FII. Elevated expression of the efflux pump AcrAB-TolC (acrA, 15.3 times) and reduced expression of outer membrane protein genes ompK35 and ompK37 (0.16 and 0.081 times, respectively) associated with various amino acid alterations on OmpK37 were observed. The presence of two carbapenemases in ST258 K. pneumoniae is of great concern due to the ability of this organism to widely disseminate.

Detection of a New cfr-Like Gene, cfr(B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program.

Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 µg/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci.

Linezolid update: stable in vitro activity following more than a decade of clinical use and summary of associated resistance mechanisms.

Linezolid, approved for clinical use since 2000, has become an important addition to the anti-Gram-positive infection armamentarium. This oxazolidinone drug has in vitro and in vivo activity against essentially all Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The in vitro activity of linezolid was well documented prior to its clinical application, and several ongoing surveillance studies demonstrated consistent and potent results during the subsequent years of clinical use. Emergence of resistance has been limited and associated with invasive procedures, deep organ involvement, presence of foreign material and mainly prolonged therapy. Non-susceptible organisms usually demonstrate alterations in the 23S rRNA target, which remain the main resistance mechanism observed in enterococci; although a few reports have described the detection of cfr-mediated resistance in Enterococcus faecalis. S. aureus isolates non-susceptible to linezolid remain rare in large surveillance studies. Most isolates harbour 23S rRNA mutations; however, cfr-carrying MRSA isolates have been observed in the United States and elsewhere. It is still uncertain whether the occurrences of such isolates are becoming more prevalent. Coagulase-negative isolates (CoNS) resistant to linezolid were uncommon following clinical approval. Surveillance data have indicated that CoNS isolates, mainly Staphylococcus epidermidis, currently account for the majority of Gram-positive organisms displaying elevated MIC results to linezolid. In addition, these isolates frequently demonstrate complex and numerous resistance mechanisms, such as alterations in the ribosomal proteins L3 and/or L4 and/or presence of cfr and/or modifications in 23S rRNA. The knowledge acquired during the past decades on this initially used oxazolidinone has been utilized for developing new candidate agents, such as tedizolid and radezolid, and as linezolid patents soon begin to expire, generic brands will certainly become available. These events will likely establish a new chapter for this successful class of antimicrobial agents.

Retrospective molecular analysis of DIM-1 metallo-ß-lactamase discovered in Pseudomonas stutzeri from India in 2000.

Among 220 clinical isolates of Gram-negative bacilli collected in India during 2000, 22 strains showing elevated imipenem MICs were evaluated for carbapenemase production. One DIM-1-producing Pseudomonas stutzeri isolate was detected, and no other carbapenemase-encoding genes were identified. This detection of a DIM-1-producing P. stutzeri isolate from India predating the finding of this gene in the index Dutch strain and the very recent detection of DIM-1 in Africa suggest an unidentified environmental source of this metallo-ß-lactamase gene.

Evaluation of clonality and carbapenem resistance mechanisms among Acinetobacter baumannii-Acinetobacter calcoaceticus complex and Enterobacteriaceae isolates collected in European and Mediterranean countries and detection of two novel ß-lactamases, GES-22 and VIM-35.

We evaluated doripenem-resistant Acinetobacter baumannii-Acinetobacter calcoaceticus complex (ACB; n = 411) and Enterobacteriaceae (n = 92) isolates collected from patients from 14 European and Mediterranean countries during 2009 to 2011 for the presence of carbapenemase-encoding genes and clonality. Following susceptibility testing, carbapenem-resistant (doripenem MIC, >2 µg/ml) isolates were screened for carbapenemases. New ß-lactamase genes were expressed in a common background and susceptibility was tested. Class 1 integrons were sequenced. Clonality was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing (Pasteur scheme). Relative expression of ß-lactam intrinsic resistance mechanisms was determined for carbapenemase-negative Enterobacteriaceae. ACB and Enterobacteriaceae displayed 58.9 and 0.9% doripenem resistance, respectively. bla(OXA-23), bla(OXA-58), and bla(OXA-24/OXA-40) were detected among 277, 77, and 29 ACB, respectively (in 8, 6, and 5 countries). Ten Turkish isolates carried bla(GES-11) or bla(GES-22). GES-22 (G243A and M169L mutations in GES-1) had an extended-spectrum ß-lactamase profile. A total of 33 clusters of ≥ 2 ACB isolates were observed, and 227 isolates belonged to sequence type 2/international clone II. Other international clones were limited to Turkey and Israel. Doripenem-resistant Enterobacteriaceae increased significantly (0.7 to 1.6%), and 15 blaKPC-2- and 22 blaKPC-3-carrying isolates, mostly belonging to clonal complexes 11 and 258, were observed. Enterobacteriaceae isolates producing OXA-48 (n = 16; in Turkey and Italy), VIM-1 (n = 10; in Greece, Poland, and Spain), VIM-26 (n = 1; in Greece), and IMP-19, VIM-4, and the novel VIM-35 (n = 1 each from Poland) were detected. VIM-35 had one substitution compared to VIM-1 (A235T) and a similar susceptibility profile. One or more resistance mechanisms were identified in 4/6 carbapenemase-negative Enterobacteriaceae. This broad evaluation confirms results from country-specific surveys and shows a highly diverse population of carbapenemase-producing ACB and Enterobacteriaceae in Europe and Mediterranean countries.

Epidemiology and carbapenem resistance mechanisms of carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries.

OBJECTIVES: To evaluate the genetic relatedness and carbapenem resistance mechanisms among carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries. METHODS: Doripenem-non-susceptible (MIC >2 mg/L) isolates were tested for susceptibility to imipenem, meropenem, doripenem, aztreonam, ceftazidime and cefepime with and without phenyl-arginine-ß-naphthylamide (PAßN) (efflux inhibitor) and/or cloxacillin (AmpC inhibitor). Carbapenemase screening was performed by PCR and sequencing. Expression of chromosomal ampC, mexA, mexC, mexE and mexX was determined by quantitative real-time PCR using P. aeruginosa PAO1 or a group of susceptible isolates as baseline. Clonality was evaluated by PFGE and multilocus sequence typing. RESULTS: Among 529 (25.6% overall) carbapenem-non-susceptible P. aeruginosa, 106 were positive for metallo-ß-lactamase (MßL) genes encoding VIM-2 (76 strains), VIM-4 (14), VIM-1 (7) and VIM-5 (5). IMP-15 and three new MßLs (IMP-33, VIM-36 and VIM-37) were detected in one strain each. An increasing prevalence of MßL producers was noted in 2011 (30.6%) compared with previous years (13.4% and 12.3% in 2009 and 2010, respectively). Isolates displayed high genetic diversity, with 401 unique profiles detected. CC235 and ST111 were detected among MßL-producing clusters. The PAßN/cloxacillin effect ranged from 90.0% to 56.5%/from 1.3% to 21.2%. OprD decrease/loss was the most prevalent intrinsic mechanism and was detected among 94.9% of the P. aeruginosa, followed by AmpC (44.4%) and MexAB-OprM (20.1%). When using the susceptible group of isolates as baseline, MexAB-OprM became as prevalent as OprD decrease/loss. CONCLUSIONS: Increasing MßL prevalence is worrisome in various European countries; however, intrinsic resistance mechanisms in a highly genetically diverse population of carbapenem-non-susceptible P. aeruginosa are probably a matter for greater concern in these countries.

Prevalence of ß-lactamase-encoding genes among Enterobacteriaceae bacteremia isolates collected in 26 U.S. hospitals: report from the SENTRY Antimicrobial Surveillance Program (2010).

Enterobacteriaceae bacteremia isolates (n = 195; 6.4% overall) collected from 26 U.S. hospitals located in 20 states were screened for various ß-lactamase classes. A total of 175 isolates carried one to eight acquired ß-lactamase genes of 44 types that were detected in 55 combinations. Eighty-five (43.6%) strains carried blaCTX-M, and blaCTX-M-15 was the most prevalent (33.8%). Genes encoding OXA-1/30 (often associated with blaCTX-M-15), CMY-2, SHV extended-spectrum ß-lactamase (ESBLs), and TEM-1 were also prevalent. Among 33 carbapenem-resistant strains, 28 carried blaKPC-2 or blaKPC-3 (17 and 11 strains, respectively), and those were recovered mostly in the New York City area (16 strains) and Houston, TX (9 strains). Fourteen new SHV variants were identified among Klebsiella pneumoniae isolates carrying one or multiple SHV alleles, three carrying G238S and/or E240K amino acid alterations that confer ESBL activity. Only two of eight K. oxytoca isolates carried acquired ß-lactamases, but most had mutations on the blaOXY promoter region, and three new OXY-encoding genes were characterized. Concordance between a commercial nucleic acid-based microarray (Check-MDR CT101) and reference methods was noted for 105/109 (97.2%) strains. Thirty-two strains having genes that are not targeted by the commercial system were detected (OXA ESBLs, PER, PSE, or intrinsic genes). Overall, a great variety of enzymes were observed, with numerous strains carrying multiple genes. Rates of CTX-M-producing strains appear to be increasing in U.S. hospitals (26.6% in 2007 to 43.8% for 2010) participating in the SENTRY Program. Furthermore, the Check-Points system seems to be a reliable, robust, and user-friendly assay for detection of enzyme-mediated resistance.

Dissemination of a pSCFS3-like cfr-carrying plasmid in Staphylococcus aureus and Staphylococcus epidermidis clinical isolates recovered from hospitals in Ohio.

Nineteen linezolid-resistant Staphylococcus epidermidis and two Staphylococcus aureus isolates recovered from two medical institutions in northeast Ohio and an S. aureus cfr index strain previously collected in the same facilities during the 2007 SENTRY Antimicrobial Surveillance Program were investigated for the genetic basis of oxazolidinone resistance and the location of cfr. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST). The location of cfr was determined by Southern blotting and hybridization. Plasmid sequencing was performed using the 454 Life Sciences (Roche) GS-FLX DNA platform. The two S. aureus isolates showed unique PFGE patterns but were multilocus sequence type 5 (ST5) and spa type t002, whereas the S. aureus index strain was ST239 and t037. Southern blot and hybridization experiments showed that cfr was plasmid located and that the S. epidermidis isolates, one of the S. aureus isolates, and the S. aureus index strain shared an identical cfr-carrying plasmid (39.3 kb). Sequencing results confirmed these findings. A 10-kb fragment containing cfr showed the highest identity (99.9%) to a 9.5-kb fragment of plasmid pSCFS3 from a bovine Staphylococcus lentus isolate from Germany. In addition, these 39.3-kb plasmids from human S. epidermidis and S. aureus exhibited BglII restriction profiles very similar to that observed for plasmid pSCFS3. The cfr-carrying plasmid detected in the remaining S. aureus isolate (7.9 kb) was distinct and showed the highest identity to the chromosomal cfr integrate found in the chromosomal DNA of a Proteus vulgaris isolate from a pig in China.

Streptococcus sanguinis isolate displaying a phenotype with cross-resistance to several rRNA-targeting agents.

This study describes a clinical case of a 71-year-old male with a history of ischemic cardiomyopathy after left ventricular assist device (LVAD) endocarditis caused by methicillin-resistant Staphylococcus epidermidis (MRSE) and a rare linezolid-resistant Streptococcus sanguinis strain (MIC, 32 µg/ml). The patient received courses of several antimicrobial agents, including linezolid for 79 days. The S. sanguinis strain had mutations in the 23S rRNA (T2211C, T2406C, G2576T, C2610T) and an amino acid substitution (N56D) in L22 and exhibited cross-resistance to ribosome-targeting agents.

Endemicity of Pseudomonas aeruginosa producing IMP-18 and/or VIM-2 MBLs from the high-risk clone ST111 in Central America.

Background: Pseudomonas aeruginosa is an important cause of serious nosocomial infections. Despite the overall genetic diversity of this species, highly conserved clonal complexes (CCs) have been observed among MDR isolates. Many of these CCs are associated with MBL-producing isolates. Objectives: To evaluate five P. aeruginosa isolates from Central America that carried IMP-18- and/or VIM-2-encoding genes from the SENTRY Antimicrobial Surveillance Program (2017-2018). Methods: Susceptibility testing was performed by broth microdilution (CLSI). WGS was performed using MiSeq (Illumina) and MinION (Oxford Nanopore). Assembled contigs from short and long reads were combined for in silico screening of resistance genes, MLST, core genome (cg)MLST and SNP analysis. Results: The P. aeruginosa isolates were collected in Panama and Mexico from patients with urinary tract infections or pneumonia. Isolates were categorized as XDR (CLSI/EUCAST). All isolates belonged to ST111 but carried different combinations of resistance-encoding genes. Transposon-associated MBL genes, blaIMP-18 and/or blaVIM-2, were chromosomally located. blaIMP-18 was detected in an In1666 integron whereas blaVIM-2 was embedded in an In59-like integron. Isolates were closely related based on cgMLST (average allele distance 2-34) and SNP analysis (5-423 different SNPs). Conclusions: MBL-producing ST111 P. aeruginosa have become endemic in Panama and may have spread to Mexico via clonal dissemination. Recombination events are apparent in the evolution of this CC. Surveillance is warranted to track the expansion and movement of this clone.

In Vitro Activity of Isavuconazole and Other Mould-Active Azoles against Aspergillus fumigatus with and without CYP51 Alterations.

Azole resistance in Aspergillus fumigatus (AFM) is mainly associated with mutations in CYP51A and its promoter region or its homologue CYP51B. We evaluated the in vitro activity of isavuconazole, itraconazole, posaconazole, and voriconazole against 660 AFM collected during 2017-2020. Isolates were tested via CLSI broth microdilution. CLSI epidemiological cutoff values were applied. Non-wildtype (NWT) isolates to azoles were screened for alterations in the CYP51 sequences using whole genome sequencing. Azoles had similar activities against 660 AFM isolates. Overall, AFM displayed WT MIC values to isavuconazole (92.7%), itraconazole (92.9%), posaconazole (97.3%), and voriconazole (96.7%). Only 66 isolates (10.0%) were NWT to 1 or more of the azoles, and 32 harbored one or more alterations in the CYP51 sequences. Of these, 29/32 (90.1%) were NWT to itraconazole, 25/32 (78.1%) were NWT to isavuconazole, 17/32 (53.1%) were NWT to voriconazole, and 11/32 (34.4%) were NWT to posaconazole. The most frequent alteration was CYP51A TR34/L98H, carried by 14 isolates. Four isolates carried the alteration I242V in CYP51A, and G448S; A9T, or G138C was carried by one isolate each. Multiple alterations in CYP51A were detected in five isolates. Alterations in CYP51B were noted in seven isolates. Among 34 NWT isolates without -CYP51 alterations, WT rates to isavuconazole, itraconazole, voriconazole, and posaconazole were 32.4%, 47.1%, 85.3%, and 82.4%, respectively. Ten different CYP51 alterations were detected in 32/66 NWT isolates. Alterations in AFM CYP51 sequences can have variable effects on the in vitro activity of the azoles that are best delineated by testing all triazoles.

A plain language summary of how some fungi samples that show resistance to antifungal medicines have changes in their genes.

WHAT IS THIS SUMMARY ABOUT?: Aspergillus fumigatus (shortened to A. fumigatus) is a fungus (plural: fungi) that can cause a serious infection in some people. A. fumigatus can become resistant to medicines known as azoles (isavuconazole, itraconazole, posaconazole, and voriconazole). This means they stop working and are not able to kill the fungus. Fungi can become resistant through changes in their genes, which are called mutations. Scientists looked at previously collected samples from people infected with A. fumigatus and found that 36 of the samples showed resistance to an azole. In 35 of these samples, scientists looked for mutations in 50 genes. These 50 genes are known to play a role in azole resistance and/or are important for fungal survival. WHAT WERE THE RESULTS?: In total, 18 out of 36 samples (50%) showed resistance to isavuconazole only. Of these, 12 had mutations in 4 genes important for fungal survival (called erg3C, erg2, erg7B and erg4B). Mutations were found in 2 genes that are the most common causes of azole resistance (called cyp51A and cyp51B). The most common mutation, called cyp51A TR34/L98H, was found in 9 samples. Of these, 8 samples showed resistance to all 4 of the azoles tested. WHAT DO THE RESULTS OF THE STUDY MEAN?: Studying mutations that make fungi resistant to medicines helps to make sure that people with fungal infections get treated with medicines that will work for them.

Molecular epidemiology of Staphylococcus epidermidis clinical isolates from U.S. hospitals.

The epidemiology of Staphylococcus epidermidis in U.S. hospitals remains limited. This study aimed to address the genetic backgrounds of linezolid-susceptible and -resistant S. epidermidis strains (isolated in 2010), including cfr-carrying strains. In addition, the antimicrobial susceptibility profiles and linezolid resistance mechanisms among clonal lineages were assessed. A total of 71 S. epidermidis isolates were selected, and linezolid-resistant strains were screened for cfr and mutations in 23S rRNA, L3, and L4. All isolates were subjected to multilocus sequence typing (MLST), and the results were analyzed by eBURST. Overall, 27 sequence types (STs) were detected, and ST5 (21.1%) and ST2 (16.9%) predominated. The majority (62/71; 87.3%) of STs belonged to clonal complex 2 (CC2), which was mostly comprised of subclusters CC2-II (41/62; 66.1%) and CC2-I (21/62; 33.9%). Other STs were grouped within CC23 or CC32 or were singletons. CC2-I strains were more likely to display a methicillin (95.2% versus 33.3 to 70.7%), a linezolid (47.6% versus 0.0 to 7.3%), or a multidrug (81.0% versus 33.3 to 36.6%) resistance phenotype. Among linezolid-resistant isolates, cfr was noted only within CC2 strains, and it was detected equally in the CC2-I (3/10; 30.0%) and CC2-II (1/3; 33.3%) subclusters. 23S rRNA mutations (G2576 [seven strains] and C2534 [one strain]) were observed only among CC2-I (8/10; 80.0%) isolates. Strains showing a G2576 alteration also had M156 (7/7; 100.0%) and/or H146 (6/7; 85.7%) L3 modifications. This study provides an overview of the S. epidermidis clonal distribution and reports higher resistance rates among CC2-I strains. The results show that cfr may be acquired and expressed by both CC2 main subclusters, while 23S rRNA mutations appeared more often within CC2-I strains. Interestingly, these 23S rRNA mutants also had L3 alterations, which may act synergistically or in a compensatory manner to minimize the fitness cost while providing survival advantages under selective pressure.

Characterization of methicillin-resistant Staphylococcus aureus strains recovered from a phase IV clinical trial for linezolid versus vancomycin for treatment of nosocomial pneumonia.

A total of 434 methicillin-resistant Staphylococcus aureus (MRSA) baseline isolates were collected from subjects enrolled in a prospective, double-blind randomized trial comparing linezolid versus vancomycin for the treatment of nosocomial pneumonia. Isolates were susceptibility tested by broth microdilution, examined for inducible clindamycin resistance by D-test, and screened for heterogeneous resistance to vancomycin (hVISA) by the Etest macromethod. All strains were subjected to Panton-Valentine leukocidin (PVL) screening, and SCCmec, pulsed-field gel electrophoresis (PFGE), and spa typing. Selected strains were evaluated by multilocus sequence typing (MLST). Clonal complexes (CCs) were assigned based on the spa and/or MLST results. Most strains were CC5 (56.0%), which originated from North America (United States) (CC5-MRSA-SCCmec II/IV; 70.0%), Asia (CC5-MRSA-II; 14.0%) and Latin America (CC5-MRSA-I/II; 12.3%). The second- and third-most-prevalent clones were CC8-MRSA-IV (23.3%) and CC239-MRSA-III (11.3%), respectively. Furthermore, the CC5-MRSA-I/II clone predominated in Asia (50.7% within this region) and Latin America (66.7%), followed by CC239-MRSA-III (32.8% and 28.9%, respectively). The European strains were CC8-MRSA-IV (34.5%), CC22-MRSA-IV (18.2%), or CC5-MRSA-I/II/IV (16.4%), while the U.S. MRSA isolates were CC5-MRSA-II/IV (64.4%) or CC8-MRSA-IV (28.8%). Among the U.S. CC8-MRSA-II/IV strains, 73.7% (56/76 [21.2% of all U.S. MRSA strains]) clustered within USA300. One strain from the United States (USA800) was intermediate to vancomycin (MIC, 4 µg/ml). All remaining strains were susceptible to linezolid, daptomycin, vancomycin, and teicoplanin. hVISA strains (14.5%) were predominantly CC5-MRSA-II, from South Korea, and belonged to a single PFGE type. Overall, each region had two predominant clones. The USA300 rate corroborates previous reports describing increased prevalence of USA300 strains causing invasive infections. The prevalence of hVISA was elevated in Asia, and these strains were associated with CC5.