Objectives: The prevalence of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases among 200 Gram-negative clinical isolates resistant to different aminoglycosides and collected worldwide during 2013 was evaluated. Methods: Selected AMEs and 16S rRNA methyltransferase genes were screened by PCR/sequencing among 49 Acinetobacter spp., 52 Pseudomonas aeruginosa and 99 Enterobacterales. Results: In total 72 isolates carried aac(6')-lb variants (36.0% overall; 55.6% Enterobacterales): 30 aac(6')lb-cr, 21 aac(6')-Ib and 21 aac(6')-Ib-like displaying substitutions L119S (alone or in combination with V71A or R173K) or S100G. Ten aph(3' )-VI variants were detected among 35 isolates (46.9% of Acinetobacter spp.). Nineteen isolates carried variants of aac(3)-I, with aac(3)-Ia (n = 13, mostly Acinetobacter spp.) being the most prevalent. Other AME genes detected were ant(3 '')-Ia (n = 41), ant(2 '')-Ia (n = 24), aac(3)-IIe (n = 23), aac(3)-IId (n = 21), aac(6')-Im (n = 13, mostly P. aeruginosa), aacA8 (n = 3), aac(3)-IIf (n =1) and aac (3)-IVa (n=1). Among 42 isolates resistant to amikacin, gentamicin and tobramycin tested for 16S rRNA methyltransferase genes, 21 (50.0%) tested positive; armA was most common (n =14), but 4 isolates carried rmtB1, 2 rmtF1 and 1 new variant rmtB4. Over 60 gene combinations, consisting of one to four AMEs and 16S rRNA methyltransferases, were observed. Cloning genes not previously characterised revealed diverse aminoglycoside resistance patterns for some AMEs, but expected results for rmtB4. Conclusions: Studies broadly evaluating these aminoglycoside resistance genes are needed. Using agents stable in the presence of these resistance genes might help overcome resistance. (C) 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Background: Echinocandins are important agents for treatment of invasive fungal infections. We evaluated the activity of CD101, a once-weekly echinocandin with extended half-life, and comparators against 606 invasive fungal isolates collected worldwide during 2014 using CLSI broth microdilution methods. Methods: 531 Candida spp. (7 species), 19 C. neoformans (CNEO) and 56 A. fumigatus (ASF) were susceptibility (S) tested for CD101, anidulafungin (ANF), caspofungin (CSF), micafungin (MCF) and azoles. CLSI clinical breakpoint (CBP) and epidemiological cutoff value (ECV) interpretive criteria were applied. Isolates displaying echinocandin MIC>ECV were sequenced for fks hot spot (HS) mutations. Results: The activity of CD101 was similar to that of other echinocandins (Table). All C. albicans (CA), C. tropicalis (CTRO), C. krusei and C. dubliniensis (n=11) were inhibited by ≤0.12 μg/ml of CD101 and were S/wild-type to other echinocandins using CBP/ECV. Five C. glabrata (CGLA) displayed CD101 MIC >0.12 μg/ml (MIC, 1-4 μg/ml), elevated CSF (2->8 μg/ml), ANF (2-4 μg/ml) and MCF (2-4 μg/ml) results and carried mutations on fks1 HS1 S629P (3 isolates/2 also had HS2 S663P), HS2 F659S (1) or S663P (3 isolates). C. parapsilosis (CPRP; n=92) and C. orthopsilosis (n=10) displayed higher MIC values (ranges 0.5-4 and 0.12-2 μg/ml, respectively), but similar results were observed for other echinocandins. Fluconazole resistance was noted among 11.0% of CGLA, 4.3% CPRP and 2.0% CA and CTRO. Echinocandins had limited activity against CNEO. CD101 activity against ASF was similar to that of MCF, two-fold greater than CSF, but less than ANF. These moulds displayed MIC values below ECVs for the mould-active azoles (itraconazole, voriconazole and posaconazole). Conclusions: CD101 was as active as other echinocandins against common fungal organisms recovered from invasive fungal infections. The extended half-life profile is very desirable for prevention and treatment of serious fungal infections, especially in patients that can then be discharged. INTRODUCTION Despite the broad utilization of echinocandins to treat invasive candidiasis (IC) in critically ill hospitalized patients, clinical resistance to these agents remains uncommon, although both breakthrough infections and acquired resistance mutations in some species of Candida have been noted. Whereas the currently available echinocandins are highly efficacious and relatively easy to use in the treatment of IC and other invasive fungal infections (IFI), they must be administered daily by intravenous infusion, potentially prolonging the hospitalization of patients undergoing therapy and limiting their use to the inpatient setting. The availability of an echinocandin with activity that is comparable to those presently in use but with a pharmacokinetic (PK) profile that allows for less frequent administration, would alter the standard-of-care therapy (e.g., echinocandin therapy) to be more easily administered in both inpatient and outpatient settings. CD101 IV is a novel echinocandin antifungal agent that displays chemical stability in plasma, aqueous solution, and at elevated temperature as well as possessing a long-acting PK. CD101 IV is being developed for once-weekly IV administration for the treatment and prevention of serious fungal infections. Less frequent administration while maintaining high exposure would alter hospital stays, improve compliance for outpatients and provide more convenient outpatient prophylaxis or maintenance treatment regimens. In the presented study, we determined the activity and potency of CD101 and comparator antifungal agents tested against 606 clinical fungal isolates collected worldwide from IFI (2014). MATERIALS AND METHODS Fungal organisms. A total of 606 non-duplicate prospectively collected fungal isolates from 38 medical centers located in North America (161 isolates; 10 sites), Europe (294; 17), the Asia-Pacific Region (82; 6) and Latin America (69; 5) were evaluated. Isolates selected were from the following sources: bloodstream, (379 strains), normally sterile body fluids, tissues or abscesses (22 strains), respiratory tract specimens (96 strains) and 109 were collected from other or non-specified body sites. Species identification. Yeast isolates were subcultured and screened using CHROMagar Candida (Becton Dickinson, Sparks, Maryland USA) to ensure purity and to differentiate Candida albicans/Candida dubliniensis, Candida tropicalis and Candida krusei. Isolates suspected to be either C. albicans or C. dubliniensis (green colonies on CHROMagar) were incubated at 45°C. All other yeast isolates were submitted to Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) using the MALDI Biotyper according to the manufacturer’s instructions (Bruker Daltonics, Billerica, Massachusetts USA). Isolates that were not identified by either phenotypic or proteomic methods were identified using sequencing-based methods as previously described. Antifungal susceptibility testing. All isolates were tested by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) methods outlined in documents M27-A3 and M38-A2. Frozen-form panels used RPMI 1640 broth supplemented with MOPS (morpholinepropane sulfonic acid) buffer and 0.2% glucose and inoculated with 0.5 to 2.5 X 103 cells/ml suspensions. MIC/MEC values were determined visually, after 24, 48 or 72 hours of incubation at 35oC, as the lowest concentration of drug that resulted in ≥50% inhibition of growth relative to the growth control or complete (100%) inhibition. CLSI clinical breakpoints were used for the five most common species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei) for echinocandins, fluconazole and voriconazole. Epidemiological cutoff values (ECV) were applied when available. Quality control was performed as recommended in CLSI documents M27-A3 and M38-A2 using strains C. krusei ATCC 6258, C. parapsilosis ATCC 22019, A. flavus ATCC 204304 and A. fumigatus MYA-3626. RESULTS • CD101 (MIC50/90, 0.03/0.06 μg/ml) inhibited all 251 C. albicans isolates at ≤0.12 μg/ml (Table 1). This compound displayed activity most similar to that of caspofungin (MIC50/90, 0.03/0.06 μg/ml). • CD101 (MIC50 and MIC90, 0.03 and 0.06 μg/ml) inhibited 95 (95.0%) of the C. glabrata isolates at ≤0.12 μg/ml (Table 1). The activity of this investigational echinocandin was two-fold greater when compared to anidulafungin or caspofungin (MIC50 and MIC90, 0.06 and 0.12 μg/ml for both compounds) and two-fold less than the activity of micafungin (MIC50 and MIC90, 0.015 and 0.03 μg/ml; Table 1). • All C. parapsilosis isolates were inhibited by CD101 (MIC50 and MIC90, 1 and 2 μg/ml) at ≤4 μg/ml (Table 1). CD101 displayed similar activity to that of micafungin (MIC50/90, 1/2 μg/ml), slightly greater activity when compared to anidulafungin (MIC50/90, 2/4 μg/ml) and was two-fold less active than caspofungin (MIC50/90, 0.5/1 μg/ml; Table 1). • C. tropicalis isolates (n=51) were considered susceptible to the clinically available echinocandins and CD101 (MIC50/90, 0.015/0.06 μg/ml) inhibited all isolates at ≤0.06 μg/ml (Table 1). • CD101 (MIC50 and MIC90, 0.03 and 0.06 μg/ml) was very active against 16 C. krusei and all isolates were inhibited at ≤0.06 μg/ml (Table 1). • The activity of CD101 (MIC50 and MIC90, 0.03 and 0.06 μg/ml; Table 1) against C. dubliniensis isolates was comparable to that of caspofungin (MIC50 and MIC90, 0.03 and 0.06 μg/ml). • CD101 (MIC50 and MIC90, 0.5 and 1 μg/ml) activity against C. orthopsilosis was similar to the activity of anidulafungin and micafungin (MIC50/90, 0.5/1 μg/ml for both). Caspofungin was two-fold more active against C. orthopsilosis isolates (MIC50 and MIC90, 0.25 and 0.5 μg/ml; Table 1) when compared to other echinocandins. • The echinocandins, including CD101, had limited activity against C. neoformans var. grubii isolates (n=19; Table 1); all isolates had MIC values at ≥8 μg/ml for these compounds. • Echinocandins displayed good activity against A. fumigatus; CD101 (MEC50 and MEC90, 0.015 and 0.015 μg/ml) activity was two-fold greater than that of caspofungin (MEC50/90, 0.03/0.03 μg/ml) and similar to that of micafungin. Anidulafungin (MEC50/90, ≤0.008/0.015 μg/ml; Table 1) was slightly more active than the other compounds from the same class. • Among the five C. glabrata isolates displaying resistant MIC results for clinically available echinocandins, one harbored a mutation on fks1 HS1 encoding alteration S629P and another two carried alterations on fks2 HS1 F659S or S663P. The two remaining isolates were collected from the same patient in Edmonton, Canada and both strains carried alterations on fks1 HS1 S629P and fks2 HS1 S663P conferring elevated caspofungin MIC results (>8 μg/ml) and MIC results of 2-4 μg/ml for CD101, anidulafungin and micafungin (Table 2). • The activity of comparator agents tested against organisms/organism groups is displayed in Figure 1. Fluconazole resistance was noted among 2.0% of C. albicans and C. tropicalis, 11.0% of C. glabrata and 4.3% of C. parapsilosis. All C. neoformans var. grubii and A. fumigatus isolates were considered wild-type for the azoles. CONCLUSIONS • The activity of CD101 tested by reference methods against common fungal species isolated from invasive infections worldwide during 2014 was comparable to currently available echinocandins. • Further evaluation of CD101 against less common species is recommended, and expanded clinical development of this long-acting echinocandin is warranted.

Background. A Phase 3 study was conducted to evaluate CAZ-AVI and “best available therapy” in the treatment of infections caused by CAZ-NS Gram-negative pathogens (NCT01644643). Here, the β-lactamase content of ENT recovered from patients was characterized. Methods. Susceptibility testing was centrally performed (CLSI). MIC criteria were pre-established for selecting ENT for screening of extended-spectrum β-lactamase (ESBL), class C β-lactamase, and/or carbapenemase genes. Isolates underwent microarray-based assay, complemented by PCR/sequencing. Relative ampC transcription levels were assessed. Results. 292 aerobic CAZ-NS ENT (MIC, ≥8 mg/mL), mostly E. coli (42.5%; 124 of 292) and K. pneumoniae (43.8%; 128 of 292) were included. Isolates were recovered from 288 mMITT patients. 4 patients had 2 species of ENT. CAZ-AVI (MIC50/MIC90, 0.5/1 mg/mL) inhibited all ENT at ≤8 mg/mL, except for 1 VIM-4 and 3 NDM-1 producers. All but 7 (117 of 124; 94.4%) E. coli harbored blaCTX-M. The other 7 isolates (7 of 124; 5.6%) had plasmid AmpC genes. 4 E. coli (4 of 117; 3.4%) had 2 blaCTX-M-like genes. 58.1% (68 of 117) blaCTX-M-harboring E. coli also had blaOXA-1/30. MeropenemNS Klebsiella spp. (meropenem MIC, ≥2 mg/mL) produced one of: KPC (6), OXA-48 (3), VIM-4 (1) or NDM-1 (1). Other Klebsiella spp. (all ESBL) were blaCTX-M-positive (116 of 120; 96.7%) or SHV producers, except for 1 OXA-9. 89 of 120 (74.2%) of blaCTX-M-carrying Klebsiella spp. had OXA enzymes (83 OXA-1/30, 2 OXA-9 and 4 OXA-10). 5 of 6 (83.3%) P. mirabilis had plasmid AmpC and 2 strains also had VIM-2 and SHV-12. One P. mirabilis carried blaCTX-M-3, blaCTX-M-15, blaOXA-9, and blaSHV-5. 10 of 19 (52.6%) Enterobacter spp. had high expression of AmpC with (5) or without CTX-M (5), while other strains had CTX-M/OXA-1/-30 (7), NMD-1 (1) or PER-2 (1). 2 blaCTX-M–positive S. marcescens were isolated. Providencia had NDM-1 (1) or ACC-4 (1). A sole M. morganii had high expression of AmpC, while C. freundii showed overexpression of AmpC (2), CTX-M (3) or a combination of both (2). One of the latter also had DHA-4. Conclusion. CAZ-AVI showed potent in vitro activity against CAZ-NS clinical trial ENT, excluding 4 MBL-producing isolates. This study demonstrates the complexity of β-lactamase genes carried by these CAZ-NS ENT. Disclosures. R. E. Mendes, AstraZeneca Pharmaceuticals: Research Contractor, Research support; M. Castanheira, AstraZeneca Pharmaceuticals: Research Contractor, Research support; L. N. Woosley, AstraZeneca Pharmaceuticals: Research Contractor, Research support; S. E. Costello, AstraZeneca Pharmaceuticals: Research Contractor, Research support; G. G. Stone, AstraZeneca Pharmaceuticals: Employee, Salary; R. K. Flamm, AstraZeneca Pharmaceuticals: Research Contractor, Research support; R. N. Jones, AstraZeneca Pharmaceuticals: Research Contractor, Research support.

ABSTRACT Ceftazidime-avibactam (MIC50/90, 0.12/0.25 μg/ml) inhibited 99.9% (20,698/20,709) of Enterobacteriaceae isolates at ≤8 μg/ml. This compound was active against resistant subsets, including ceftazidime-nonsusceptible Enterobacter cloacae (MIC50/90, 0.25/0.5 μg/ml) and extended-spectrum β-lactamase (ESBL) phenotype isolates. An ESBL phenotype was noted among 12.4% (1,696/13,692 isolates from targeted species) of the isolates, including 776 Escherichia coli (12.0% for this species; MIC50/90, 0.12/0.25 μg/ml), 721 Klebsiella pneumoniae (16.3%; MIC50/90, 0.12/0.25 μg/ml), 119 Klebsiella oxytoca (10.3%; MIC50/90, 0.06/0.25 μg/ml), and 80 Proteus mirabilis (4.9%; MIC50/90, 0.06/0.12 μg/ml) isolates. The most common enzymes detected among ESBL phenotype isolates from 2013 (n = 743) screened using a microarray-based assay were CTX-M-15-like (n = 307), KPC (n = 120), SHV ESBLs (n = 118), and CTX-M-14-like (n = 110). KPC producers were highly resistant to comparators, and ceftazidime-avibactam (MIC50/90, 0.5/2 μg/ml) and tigecycline (MIC50/90, 0.5/1 μg/ml; 98.3% susceptible) were the most active agents against these strains. Meropenem (MIC50/90, ≤0.06/≤0.06 μg/ml) and ceftazidime-avibactam (MIC50/90, 0.12/0.25 μg/ml) were active against CTX-M-producing isolates. Other enzymes were also observed, and ceftazidime-avibactam displayed good activity against the isolates producing less common enzymes. Among 11 isolates displaying ceftazidime-avibactam MIC values of >8 μg/ml, three were K. pneumoniae strains producing metallo-β-lactamases (all ceftazidime-avibactam MICs, >32 μg/ml), with two NDM-1 producers and one K. pneumoniae strain carrying the blaKPC-2 and blaVIM-4 genes. Therapeutic options for isolates producing β-lactamases may be limited, and ceftazidime-avibactam, which displayed good activity against strains, including those producing KPC enzymes, merits further study in infections where such organisms occur.

The aim of the study was to characterize forty-eight Acinetobacter baumannii (ACB) isolates with confirmed tigecycline MIC values >2mg/L observed in six Latin American (LATAM) hospitals (four countries) in 2011. During 2005-2011, 6,923 ACB isolates were collected as part of the SENTRY Program, and tigecycline susceptibility was quantified using the reference broth microdilution method. A total of 102/1881 ACB from LATAM hospitals displayed tigecycline minimum inhibitory concentration (MIC) values >2mg/L, showing an increase from 4.3% in 2010 to 10.5% in 2011, which is considerably high when compared to other geographical regions. Forty-eight ACB from 2011 displaying elevated tigecycline MICs were typed by pulsed-field gel electrophoresis, which showed multiple clusters in Sao Paulo, Brazil, and a major clone in Guadalajara, Mexico. Eighteen unique isolates had the expression of adeA and adeF determined and results compared to a group of tigecycline-susceptible strains, which demonstrated that 18/18 strains had significantly increased expression of AdeABC and three isolates overexpressed AdeFGH. Sequencing of adeS and adeR revealed that 11 isolates displayed adeS mutations, and 5 isolates had mutations in adeR. Sequencing of trm showed frameshift mutations in eight isolates and insertion sequences leading to nonfunctional proteins in three isolates. TetX-encoding genes were not detected. We documented the recent increase of ACB displaying elevated tigecycline MICs in LATAM hospitals, dominantly due to the clonal expansion of isolates in Brazil and Mexico. Control of tigecycline usage in those countries and more strict infection control practices in the involved hospitals should be considered to reduce such outbreaks.

ABSTRACT 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 β-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 β-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 β-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 bla KPC-2 - and 22 bla KPC-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.