MethodsMinimum Inhibitory Concentrations Research Articles

Emergence of novel hypervirulent Acinetobacter baumannii strain and herpes simplex type 1 virus in a case of community-acquired pneumonia in China

BackgroundA. baumannii is an important and common clinical pathogen, especially in the intensive care unit (ICU). This study aimed to characterize one hypervirulent A. baumannii strain in a patient with community-acquired pneumonia and herpes simplex type 1 virus infection. MethodsMinimum inhibitory concentrations (MICs) were determined using the Kirby-Bauer (K-B) and broth microdilution methods. Galleria mellonella infection model experiment was conducted. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The resistance and virulence determinants were identified using the ABRicate program with ResFinder and the VFDB database. The capsular polysaccharide locus (K locus) and lipooligosaccharide outer core locus (OC locus) were identified using Kleborate with Kaptive. Phylogenetic analyses were conducted using the BacWGSTdb server. ResultsA. baumannii XH2146 strain belongs to ST10Pas and ST447Oxf. The strain was resistant to cefazolin, ciprofloxacin, and trimethoprim/sulfamethoxazole (TMP-SMX). Bautype and Kaptive analyses showed that XH2146 contains OCL2 and KL49. WGS analysis revealed that the strain harbored blaADC-76, blaOXA-68, ant(3′’)-IIa, tet(B), and sul2. Notably, tet(B) and sul2, both were located within a 114,700-bp plasmid (designated pXH2146–1). Virulence assay revealed A. baumannii XH2146 possessed higher virulence than A. baumannii AB5075 at 12 h. Comparative genomic analysis showed that A. baumannii ST447 strains were mainly isolated from the USA and exhibited a relatively close genetic relationship. Importantly, 11 strains were observed to carry blaOXA-58; blaOXA-23 was identified in 11 isolates and three ST447 A. baumannii strains harbored blaNDM-1. ConclusionsEarly detection of community-acquired hypervirulent Acinetobacter baumannii strains is recommended to prevent their extensive spread in hospitals.

Antimicrobial activity of eravacycline and other comparative agents on aerobic and anaerobic bacterial pathogens in Taiwan: A clinical microbiological study

ObjectivesEravacycline, a new tetracycline derivative, exhibits broad-spectrum antimicrobial susceptibility. This study aimed to comprehensively investigate in vitro activities of eravacycline, tigecycline, and ertapenem against various Gram-positive, Gram-negative, and anaerobic bacteria. MethodsMinimum inhibitory concentrations (MICs) were determined using the broth microdilution method. The following bacterial species were collected: vancomycin-sensitive (VS) Enterococci species, vancomycin-resistant Enterococci species (VRE), Staphylococcus aureus, Streptococcus anginosus, Bacteroides species, Clostridioides difficile, Clostridium innocuum, Clostridium perfringens, Parabacteroides distasonis, and Stenotrophomonas maltophilia. ResultsWe found that eravacycline exhibited superior in vitro activity compared to tigecycline and ertapenem. Notably, it exhibited the lowest MIC90 for several bacterial species, including VS E. faecalis (0.12 µg/mL), VS E. faecium (0.12 µg/mL), and others. Besides, VRE was susceptible to eravacycline (MIC90:0.12 µg/mL) and tigecycline (MIC90:0.12 µg/mL), but was all resistant to ertapenem (MIC90 > 64 µg/mL). S. aureus was also susceptible to eravacycline (MIC90:0.5 µg/mL) as well as tigecycline (MIC90:1.0 µg/mL). Furthermore, S. anginosus showed higher susceptibility to eravacycline (MIC90:2.0 µg/mL) and tigecycline (MIC90:4.0 µg/mL), but lower to ertapenem (MIC90:32.0 µg/mL). Eravacycline and tigecycline also demonstrated good susceptibility to anaerobes, including Bacteroides species (susceptibility rate: 100%), P. distasonis (100%), C. difficile (94.1‒100%), C. innocuum (94.1‒96.1%), and C. perfringens (88.9‒96.3%). For S. maltophilia, both tigecycline and eravacycline showed an MIC90 of 2 µg/mL. A moderate-to-strong correlation (rho = 0.608–0.804, P < 0.001) was noted between the MIC values of eravacycline and tigecycline against various bacterial species. ConclusionsOur study highlights the potential of eravacycline as an effective treatment option for multidrug-resistant bacterial infections.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents

ObjectivesWe recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. MethodsMinimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. ResultsADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. ConclusionOur findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

In Vitro Susceptibility of Burkholderia pseudomallei Isolates to Cefiderocol and Ceftazidime/Avibactam from Odisha, India.

Introduction and Objectives The availability of a limited arsenal of antibacterial agents effective against Burkholderia pseudomallei, the causative agent of melioidosis, together with sporadic reports of emergence of resistance necessitates an evaluation of in vitro activity of new antimicrobials against clinical B. pseudomallei isolates. Cefiderocol (CFDC), a novel siderophore cephalosporin, and ceftazidime-avibactam (CZA), a new β lactam combination agent, have shown promising results for the treatment of difficult-to-treat Gram-negative bacilli infections with limited treatment options. This study was conducted to determine the in vitro activity of CFDC and CZA against a contemporary collection of 60 B. pseudomallei clinical isolates. Materials and Methods Minimum inhibitory concentrations (MIC) of CFDC and CZA were determined by broth microdilution and E-test, respectively. The performance of disk diffusion was also evaluated for CFDC. Results All B. pseudomallei isolates were susceptible to CFDC and CZA with MIC range of 0.125 to 2 mg/L and 0.19 to 1 mg/L, respectively. Zone diameters for CFDC ranged from 31 to 40 mm. Conclusion CFDC and CZA exhibited excellent in vitro activity against 60 B. pseudomallei isolates. Further pharmacokinetic-pharmacodynamics studies and clinical trials are needed to prove the clinical efficacy of CFDC and CZA in the treatment of melioidosis.

Activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa from patients in the Middle East and Africa – Study for Monitoring Antimicrobial Resistance Trends (SMART) 2017-2020

ObjectivesWe evaluated the activity of ceftolozane/tazobactam (C/T), and comparators against clinical Pseudomonas aeruginosa isolates collected for the global Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance program in ten countries in the Middle East and Africa to augment scarce standardized surveillance data in this region. MethodsMinimum inhibitory concentrations (MICs) were determined using Clinical and Laboratory Standards Institute broth microdilution and interpreted with European Committee on Antimicrobial Susceptibility Testing breakpoints. P. aeruginosa isolates testing with C/T MIC >4 mg/l or imipenem MIC >2 mg/l were screened for β-lactamase genes. ResultsC/T was active against 91.4% and 87.0% of P. aeruginosa isolates from the Middle East and Africa, respectively (14-21 and 7-16 percentage points higher than most β-lactam comparators, respectively). Considerable variation in susceptibility was seen across countries, which largely correlated with the observed prevalence of carbapenemases and/or extended-spectrum β-lactamases. Differences across countries were smaller for C/T than for the β-lactam comparators, ranging from 81% C/T-susceptible among isolates from Jordan to 95% for Qatar. Among subsets resistant to meropenem, ceftazidime, or piperacillin/tazobactam, C/T maintained activity against 51-73% of isolates from the Middle East and against 27-54% from Africa (where metallo-β-lactamase and GES carbapenemase rates were higher). ConclusionGiven the desirability of β-lactam use among clinicians, C/T represents an important option in the treatment of infections caused by P. aeruginosa.

Tafenoquine exhibits broad spectrum antifungal activity at clinically relevant concentrations in vitro and decreases lung fungal burden in an invasive pulmonary model of Rhizopus in vivo

BackgroundTafenoquine is active against a broad range of pathogens and accumulates extensively in the lung. We profiled the susceptibility of fungal pathogens to tafenoquine in vitro and in vivo. MethodsMinimum inhibitory concentrations [MICs] of medically important fungal pathogens were determined using conventional in vitro assays. The daily maximum tolerated dose [MTD] of tafenoquine was determined in neutropenic mice and the effect of two doses of tafenoquine [MTD and 0.5xMTD] on survival and fungal burden were assessed in Rhizopus and Aspergillus lung infections models. ResultsMean MICS against panels of yeasts and dimorphic/filamentous fungi were 4.5 and 8.3 ug/mL. The MTD of tafenoquine was 5 mg/kg/day. Against Aspergillus [MIC 16 ug/mL], tafenoquine did not increase survival or decrease fungal burden. Against Rhizopus, [MIC 4 ug/mL], tafenoquine decreased lung fungal burden [by 0.5 logs, P < 0.05 at the MTD] in a dose-related manner. Survival in the high-dose [MTD] tafenoquine group was 30% whereas it was 0% in the vehicle group and in most legacy studies. ConclusionsTafenoquine exhibited broad spectrum activity against medically important yeasts and fungi in vitro and a dose-related antifungal effect in a Rhizopus lung infection model at clinically relevant doses.

Cashew nut shell liquids: Antimicrobial compounds in prevention and control of the oral biofilms

ObjectiveThe aim was to evaluate the antibacterial and antibiofilm activity of natural (n-CNSL) and technical (t-CNSL) cashew nut shell liquid against streptococci and enterococci related to dental caries and chronic apical periodontitis, respectively. Material and methodsMinimum inhibitory concentrations (MIC) and minimal bactericidal concentration (MBC) were determined to assess the antimicrobial effect of both CNSLs (n-CSNL and t-CNSL) against S. oralis ATCC 10557, S. sobrinus ATCC 6715, S. parasanguinis ATCC 903, S. mutans UA 159 and E. faecalis ATCC 19433. The antibiofilm activity was evaluated by total biomass quantification, colony forming unit (CFU) counting and scanning electron microscopy (SEM). Furthermore, cytotoxic effect of the substances was evaluated on L929 and HaCat cell lines by MTS assay. ResultsThe n-CNSL and t-CNSL showed inhibitory and bactericidal effect against all strains tested in this study, with MIC and MBC values ranging from 1.5 to 25 μg/mL. Overall, both CNSLs showed significant reduction in biomass quantification and enumeration of biofilm-entrapped cells for the strains analyzed, in biofilm formation and preformed biofilms (p < 0.05). In biofilm inhibition assay, the t-CNSL and n-CNSL showed reduction in biomass and CFU number for all bacteria, except in cell viability of S. parasanguinis treated with t-CNSL (p > 0.05). Indeed, SEM images showed a reduction in the amount of biomass, bacterial cells and changes in cellular morphology of S. mutans. ConclusionIn conclusion, both substances showed effective antibacterial and antibiofilm activity against the strains used in the study, except in viability of S. parasanguinis cells treated with t-CNSL.

Accuracy of gradient diffusion method for susceptibility testing of dalbavancin and comparators

ABSTRACT Objectives This multicenter study aimed to assess the performances of gradient diffusion (GD) method in comparison to broth microdilution (BMD) method for susceptibility testing of dalbavancin, daptomycin, vancomycin, and teicoplanin. Methods Minimum Inhibitory Concentrations (MICs) were retrospectively determined concomitantly by BMD and GD methods, for 93 staphylococci and enterococci isolated from clinical samples. BMD was considered as the gold standard. Essential (EA) and categorical agreements (CA) were calculated. Discordant categorical results were categorized as major (ME) and very major errors (VME). Results EA and CA were 95.7% and 96.8%, 82.8% and 100%, 97.8% and 96.8%, and 94.6% and 95.7% for dalbavancin, daptomycin, vancomycin, and teicoplanin respectively. Concerning dalbavancin, 3 ME without any VME were observed and discrepancies were low (≤ to 2 two-fold dilutions) between both methods. VME were noted in 1 and 3 cases for vancomycin and teicoplanin, respectively, and resulted from 1 two-fold dilution discrepancy in each case. EA was lower for daptomycin. When they were discrepant, BMD MICs were systematically higher than GD ones. Nevertheless, no categorical discrepancy was noted. Conclusions GD appears as an acceptable and convenient alternative for dalbavancin, vancomycin, and teicoplanin MICs determination. Our study also emphasizes how achieving accurate daptomycin MICs remains challenging.

Antimicrobial susceptibility pattern of oral gram negative anaerobes from Indian subjects

ObjectivesThere is paucity of information on the antimicrobial susceptibility pattern of oral anaerobic bacteria. In this study, an attempt has been made to evaluate the antimicrobial susceptibility/resistance trend of oral Gram negative bacteria from Indian subjects. MethodsMinimum inhibitory concentrations (MIC) of 304 isolates against twelve different antibiotics were determined using gradient diffusion MIC strips. The organisms were isolated and identified based on phenotypic characteristics and included Porphyromonas gingivalis, Prevotella species, Tannerella forsythia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcoitans, Eickenella corrodens and Capnocytophaga species. For each antimicrobial agent, MIC50 and MIC90 were calculated and expressed. ResultsResistance to azithromycin, clindamycin, and amoxicillin was observed in most of the anaerobic bacterial species studied. High degree of susceptibility was observed to amoxillin-clavulanic acid, doxycycline and moxifloxacin. A single strain of P. melaninogenica was resistant to moxifloxacin. The susceptibility pattern varied with cephalosporins among species. Ceftriaxone showed highest and cefazolin least efficacy among cephalosporins. All anaerobic bacteria tested were susceptible to metronidazole. Strains of T. forsythia were more resistant to several antibiotics than other anaerobic bacteria. All three species of capnophilic bacteria displayed high degree of resistance to metronidazole and significant resistance to amoxicillin, azithromycin, clindamycin, cefazolin and cefuroxime. ConclusionsAmoxicillin-clavulanic acid, doxycycline, moxifloxacin and metronidazole appeared to be the most effective drugs against gram negative anaerobic bacteria. However, the MIC50 and MIC90 values against metronidazole were on the higher side of the normal indicating a potential for developing resistance.

Photodynamic therapy: A treatment option for terbinafine resistant Trichophyton species.

Terbinafine is a first-line agent against Trichophyton-infections. However, treatment failure and resistance due to squalene epoxidase (SQLE) alterations are increasingly being reported. Photodynamic therapy (PDT) is based on combining a photosensitizer, light and oxygen to create photo-activated reactive oxygen species. It has demonstrated in vitro and in vivo activity against various microorganisms including dermatophytes. We investigated if PDT is equally effective against terbinafine resistant and susceptible strains. Minimum inhibitory concentrations (MIC) of methylene blue (MB)-PDT against wildtype and resistant Trichophyton rubrum and Trichophyton interdigitale were determined in duplicate in microtitre plates following EUCAST E.Def 11.0 reference methodology. Included mutants harboured F397L, L393F, L393S, F415S or F397I SQLE-alterations. Illumination with red diode light was performed after <3 min, 30 min and 3 h of incubation, respectively, and plates were cultured at 25 °C for 5 days. Geometric mean MICs and MIC ranges were calculated for each isolate. MB-PDT led to complete inhibition of all isolates at geometric mean concentrations of 1-16 mg/L. Efficacy was independent of incubation time prior to illumination, terbinafine susceptibility (MICs ≤0.004-4 mg/L) and presence of SQLE mutations. However, the MB-PDT MIC was slightly elevated (MB: 2-8 mg/L and 8-16 mg/L) in isolates from two pigmented cultures of Trichophyton interdigitale (one wildtype and one harbouring L393F) with a darker color when compared to unpigmented cultures (MB: 0.5-4 mg/L). Terbinafine resistant and susceptible strains are equally susceptible to MB-PDT. Lower efficacy was observed against dark coloured isolates which we speculate may be due to melanisation interfering with photo-activation due to preferential light absorption.

1572. Combination Cefuroxime and Sulopenem is active in vitro against Mycobacterium abscessus

Background Mycobacterium abscessus (Mab) is a highly drug-resistant nontuberculous mycobacteria (NTM). Efforts to discover new treatments for Mab infections are accelerating with a focus on cell wall synthesis proteins (L, D-transpeptidases, LdtMab1-5, and D, D-carboxypeptidase) that are targeted by combination β-lactam antibiotics. The US Food and Drug Administration (FDA) has granted Qualified Infectious Disease Product (QIDP) to the oral and intravenous (IV) formulations of Sulopenem (SUL). Data on SUL in vitro activity against Mab is currently unavailable. Here, we evaluated activity of SUL alone and in combination with Cefuroxime salt (CEF) against representative clinical isolates belonging to the Mab complex. Both CEF and SUL are available in oral formulation and can be considered as oral step-down therapy.MethodsMinimum inhibitory concentrations (MICs) of SUL and CEF alone and in combination were determined using microdilution. Approximately 5 x 105 colony-forming units (CFU) per milliliter were inoculated into Middlebrook 7H9 Broth supplemented with 10% (vol/vol) oleic albumin dextrose catalase and 0.05% (vol/vol) Tween 80. CEF was added at fixed concentration of 4 µg/ml to serial dilutions of SUL. Mab isolates were incubated with test agents at 30 °C for 48 h, and MIC was defined as lowest antibiotic concentration that prevented visible bacterial growth.ResultsFifty-five clinically derived and previously characterized isolates were tested in these assays. MIC50 and MIC90 of CEF is 16 and 32 ug/ml; MIC50 and MIC90 of SUL is 2 and 4 ug/ml, the range of MICs are as follows: CEF (8 → 64 ug/ml); SUL (1→8 ug/ml); and SUL and CEF at fixed 4 ug/ml (< 0.25 → 4 ug/ml). Combination SUL and CEF lowered MIC to < 0.25 ug/ml in 52 clinical isolate (Figure).Fig. MIC distributions of cefuroxime salt, sulopenem, sulopenem with 4 μg/ml cefuroxime monohydrate against 55 Mab clinical strains ConclusionOur results support the emerging hypothesis that dual β-lactam therapy is a promising strategy in the treatment of serious Mab infections. Investigating the biochemical rationale for this combination will support the application to clinical trials.DisclosuresRobert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

810. Resistance to the Minocycline-Rifampin-Chlorhexidine (MRCH) combination does not emerge in biofilms of Tetracycline and Rifampin resistant bacteria grown on MRCH catheters depleted below antimicrobially effective concentrations

BackgroundCentral Line Associated Bloodstream Infections (CLABSIs) remain a significant medical problem for critically ill cancer patients who required catheters for extended durations. Minocycline (M) -Rifampin (R) loaded catheters have shown the greatest impact on reducing CLABSIs; however, there is a risk for developing antibiotic resistant organisms when exposed to catheters whose concentration becomes depleted below antimicrobially effective levels due to extended indwells. Chlorhexidine (CH) and M-R combination catheters (MRCH) have been proposed as a next generation catheter with improved performance. Here we studied whether bacteria that were Tetracycline and Rifampin resistant became resistant to MRCH when allowed to form biofilms on MRCH catheters depleted below antimicrobially effective MRCH concentrations.MethodsMinimum inhibitory concentrations (MICs) of Tetracycline and/or Rifampin resistant stock isolates were measured by standard microbroth dilution methods. MRCH catheters were depleted to below antimicrobially effective concentrations by soaking in serum for 6 weeks. The resistant bacteria were then allowed to form biofilm for 24 hrs on the depleted catheters in broth. Following 24 hour incubation the adherent (breakthrough) bacteria were removed by sonication and MICs were remeasured. The same organisms grown on non-antimicrobial catheters were used as controls.ResultsMICs (ug/mL) of the organisms against each agent and the combination are tabulated below:MICs (ug/mL) of the organisms against each agent and the combination ConclusionThe M and R resistant bacteria did not develop in vitro resistance to the MRCH combination after forming biofilms on MRCH catheters depleted below antimicrobially effective concentrations.Disclosures Joel Rosenblatt, PhD, Cook Medical (Shareholder, Other Financial or Material Support, Inventor of the MRCH catheter technology which is owned by the University of Texas MD Anderson Cancer Center and has been licensed to Cook Medical)Novel Anti-Infective Technologies (Shareholder, Other Financial or Material Support, Inventor of the MRCH catheter technology which is owned by the University of Texas MD Anderson Cancer Center and has been licensed to Cook Medical) Issam I. Raad, MD, Citius (Other Financial or Material Support, Ownership interest)Cook Medical (Grant/Research Support)Inventive Protocol (Other Financial or Material Support, Ownership interest)Novel Anti-Infective Technologies (Shareholder, Other Financial or Material Support, Ownership interest)

1256. In Vivo Activity and Structural Characterization of a New Generation γ-Lactam Siderophore Antibiotic Against Multidrug-Resistant Gram-Negative Bacteria and Acinetobacter spp

BackgroundMultidrug-resistant (MDR) A. baumannii presents a critical need for innovative antibacterial development. We have identified a new series of γ-lactam (oxopyrazole) antibiotics that target penicillin binding proteins (PBPs) and incorporate a siderophore moiety to facilitate periplasmic uptake. YU253911, an advanced iteration of this class shows potent in vitro activity against clinically relevant Gram-negative organisms including Acinetobacter spp.MethodsMinimum inhibitory concentrations (MICs) for YU253911 were determined using broth microdilution against a 198-member panel of clinical isolates of Acinetobacter spp. Resistant strains were further evaluated for susceptibility to YU253911 in combination with sulbactam. The antibiotic’s target protein was evaluated by binding studies with Bocillin™, a fluorescent penicillin analogue, and modeled in the PBP active site. YU253911 was evaluated in vivo in a mouse soft tissue infection model.ResultsMIC testing for YU253911 revealed an MIC50 of 0.5 μg/mL and an MIC90 of 16 μg/mL, which compared favorably to all tested β-lactam antibiotics including penicillins, cephalosporins, monobactams and carbapenems (MIC50 = 2 to > 16 μg/mL). Combination with sulbactam augmented the activity of the agent. There was no apparent correlation between YU253911-resistance and the presence of specific β-lactamase genes, and incubation with representative β-lactamase proteins (KPC-2, OXA-23, OXA-24, PER-2, PDC-3, NDM-1, VIM-2, and IMP-1) showed negligible hydrolysis of the agent. YU253911 showed promising preclinical pharmacokinetics in mice with a 15 h half-life from intravenous administration and demonstrated a dose-dependent reduction in colony forming units from 50 and 100 mg/kg q6h dosing in a mouse thigh infection model using P. aeruginosa.ConclusionYU253911, a new generation γ-lactam antibiotic effective against MDR A. baumannii demonstrated promising in in vitro potency and favorable pharmacokinetics which correlated with in vivo efficacy.Disclosures Krisztina M. Papp-Wallce, PhD, Entasis (Grant/Research Support)Merck (Grant/Research Support)Venatorx (Grant/Research Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Antimicrobial susceptibility of Neisseria gonorrhoeae in Barcelona during a five-year period, 2013 to 2017.

IntroductionIncreasing rates of antimicrobial resistance in Neisseria gonorrhoeae cause problems for treating gonorrhoea.AimThis observational study aimed to describe isolates from all patients found infected with N. gonorrhoeae, in Barcelona, Spain, between 2013 and 2017, and with available antimicrobial susceptibility data.MethodsMinimum inhibitory concentrations (MICs) of penicillin (PEN), cefixime (CFM), ceftriaxone (CRO), azithromycin (AZM), ciprofloxacin (CIP), spectinomycin (SPT), fosfomycin (FOF) and gentamicin (GEN) were determined by E-test. Susceptibility was assessed using clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing. Time trends for PEN, CFM, AZM and CIP were investigated using logistic regression.ResultsOf 1,979 patients with infection (2,036 isolates), 1,888 (95.4%) were men. Patient median age was 32 years. The proportions of isolates resistant to extended-spectrum cephalosporins were low, with 0.3% (5/1,982) resistant to CRO and 4.9% (98/1,985) to CFM. AZM resistance prevalence was 2.7% (52/1,981), including 16 isolates detected in 2016 and 2017, with high-level resistance. For CIP, 51.3% (1,018/1,986) of isolates were resistant, and for PEN, 20.1% (399/1,985). All isolates were susceptible to SPT. MIC50 and MIC90 values of GEN were 4 and 6 mg/L and of FOF 12 and 24 mg/L, respectively. Between 2013 and 2017, PEN and CFM resistance rates each decreased from 28.1% (92/327) to 12.2% (70/572) and from 8.3% (27/327) to 4.4% (25/572) (p ≤ 0.0073). In contrast, AZM resistance prevalence appeared to increase from 1.5% in 2014 (5/340) to 3.0% (17/572) in 2017. No trend was identified for CIP.ConclusionAntimicrobial susceptibility surveillance is important to timely detect new phenotypes and trends.

First reported genome of an mcr-9-mediated colistin-resistant Salmonella Typhimurium isolate from Brazilian livestock

ObjectivesTo investigate the genetic context of colistin resistance in anmcr-9-harbouring Salmonella Typhimurium ST19 strain from swine in Brazil. MethodsMinimum inhibitory concentrations (MIC) to colistin were determined by broth microdilution. Whole-genome sequencing was performed on an Illumina MiSeq system, followed by de novo genome assembly using SPAdes 1.13.1. The draft genome sequence was annotated in Prokka using KBase online server. Downstream analyses for resistome and plasmid detection were performed using online tools available at the Center for Genomic Epidemiology. The strain was typed in silico using MLST 2.0. Phylogenetic analysis involving 24 other genomes ofSalmonella Typhimurium ST19 and mcr-9-harbouring Salmonella Typhimurium isolated from humans, livestock and foodstuff in different regions was also performed. ResultsAssembly of the draft genome resulted in 5245 protein-coding sequences, 14 rRNAs, 83 tRNAs and a GC content of 51.81%. The strain was identified asSalmonella Typhimurium ST19 harbouring a 265.5-kb pN1566-2 plasmid carrying genes encoding resistance to colistin (mcr-9.1), aminoglycosides (aadA1), tetracycline [tet(C)] and sulfonamides (sul1). Our findings indicate that the Salmonella Typhimurium ST19 strain in this study showed low genetic variability compared with Salmonella Typhimurium ST19 isolated from swine and poultry in Brazil, and was less related to those reported in other countries. ConclusionsThis is the first reported genome of a phenotypically colistin-resistantSalmonella Typhimurium harbouring the mcr-9 variant in Brazilian livestock. This genome will aid global investigations on epidemiological and evolutionary aspects of plasmid-mediated colistin resistance and the role of colistin-resistant Salmonella Typhimurium ST19 lineage as a zoonotic pathogen.

1385. Mechanism-Based, In Vitro Inhibition of Mycobacterium abscessus: Assessing β-Lactam Therapy

Background M. abscessus (Mab) is an emerging pathogen, a highly drug-resistant rapidly-growing nontuberculous mycobacteria. Mab L, D transpeptidases (LdtMab 1–5), D,D carboxypeptidase and BlaMab β-lactamase are important targets. Herein, we tested the susceptibility of ceftaroline (TAR) and imipenem (IMI) alone and in combinations with two diazabicyclooctanone β-lactamase inhibitors (BLI), relebactam (REL) and avibactam (AVI), against representative clinical isolates belonging to the Mab complex and assessed the mechanism of inhibition using mass spectrometry (QTOF-MS)MethodsMinimum inhibitory concentrations (MICs) of TAR and IMI with or without AVI and REL and a TAR-IMI combination with and without REL were determined using microdilution. Approximately 5 x 105 colony-forming units (CFU) per milliliter were inoculated into Middlebrook 7H9 broth supplemented with 10% (vol/vol) oleic albumin dextrose catalase and 0.05% (vol/vol) Tween 80. AVI or REL were added at fixed concentration of 4 µg/mL to serial dilutions of TAR or IMI. For the TAR-IMI combinations, IMI at 1 µg/mL, and serial dilutions of TAR were used. Mab isolates were incubated with test agents at 30°C for 48 h, and MIC was defined as lowest antibiotic concentration that prevented visible bacterial growth. (QTOF-MS) was used to assess intermediates of BlaMab, LdtMab1 and LdtMab2 with TAR, IMI, AVI, and RELResults In-vitro susceptibility testing on representative clinical Mab strains (table). MIC90 was > 128 μg/mL for TAR and 8 μg/mL for IMI. Combination of TAR and IMI lowered MIC’s of all clinical isolates to <0.06 μg/mL. Addition of REL or AVI lowered TAR MICs but had minimal or no impact on IMI or TAR-IMI MICs. Mass spectrometry analyses of BlaMab, LdtMab (1–2) alone and incubated with IMI, TAR, REL and AVI (figure). BlaMab β-lactamase bound the AVI and REL, but acyl complexes with TAR or IMI were not detected. LdtMab (1–2) form stable acyl complexes with AVI, REL, TAR, and IMI.ConclusionAddition of IMI to TAR lowers MICs of TAR against Mab to therapeutically achievable concentrations. It would be welcome news for clinicians who are treating patients with highly resistant Mab infection that the combination of TAR and IMI is commercially available and thus might be considered as part of a rescue regimen. Disclosures All authors: No reported disclosures.

725. WCK 5222 (Cefepime/Zidebactam): An In Vitro Assessment of Activity Compared with Current Dual-Antibiotic Options Against Multidrug-Resistant Pseudomonas aeruginosa

Background Pseudomonas aeruginosa (PSA) is an opportunistic pathogen known to cause complications in critically ill patients worldwide. In those at risk of infection with multidrug-resistant strains (MDR-PSA), dual antibiotic therapy is often considered. However, this practice may contribute to rising resistance rates and poor outcomes if empirical selection is suboptimal. WCK 5222 (cefepime/zidebactam), a novel β-lactam/β-lactam enhancer, may offer a solution.MethodsMinimum inhibitory concentrations (MICs) were determined for WCK 5222, amikacin (AMK), fosfomycin (FOF), cefepime (FEP), ceftolozane/tazobactam (C/T), and meropenem (MEM) against 18 clinical PSA isolates using gradient diffusion strip (GDS) methods. Activities of FEP, C/T, and MEM in combination with AMK and FOF were assessed using GDS for isolates nonsusceptible to the β-lactam (MICs >8 mg/L, >4/4 mg/L, and >2 mg/L, respectively). Synergy was defined as a fractional inhibitory concentration index ≤ 0.5. Instances of restored β-lactam susceptibility when tested in combination were compared with the proportion of WCK 5222 MICs ≤ 8 mg/L.ResultsWCK 5222 MICs ranged from 2 to 32 mg/L (MIC50, 8 mg/L). Rates of susceptibility were: AMK (67%), FOF (44%, MIC ≤ 64 mg/L), FEP (6%), C/T (33%), MEM (0%). Combinations with C/T most frequently demonstrated synergy (C/T-FOF, 42%; C/T-AMK, 33%) and restored C/T susceptibility was observed in 42% of assessments with FOF and in 50% with AMK. For FEP combinations, synergy was observed in 29% and 18% of assessments with FOF and AMK, respectively, with restored susceptibility in 6% for both combinations. Synergy occurred in 11% and 6% of assessments of MEM with FOF and AMK, respectively, with zero instances of restored susceptibility. In total, β-lactam susceptibility was restored in 14% (13/94) of combinations compared with 78% (14/18) of WCK 5222 MICs ≤ 8 mg/L.ConclusionIn a selection of MDR-PSA isolates that included carbapenem- and C/T-resistant strains, WCK 5222 MICs ≤ 8 mg/L (cefepime susceptible) were observed more frequently than restoration of susceptibility in select β-lactams in combination with FOF or AMK. WCK 5222 monotherapy may offer enhanced coverage of MDR-PSA over empirically selected combination therapies.Disclosures All authors: No reported disclosures.

685. An In Vitro Investigation of WCK 5222 (Cefepime/Zidebactam) and Currently Available Combination Antibiotic Regimens Against Enterobacteriaceae That Co-express Serine-β-Lactamase (SBL) and Metallo-β-Lactamase (MBL) Enzymes

BackgroundCarbapenem-resistant Enterobacteriaceae (CRE) that simultaneously harbor SBLs and MBLs may demonstrate pan-drug resistance. Current therapeutic options include combinations of aztreonam (ATM), which is resistant to hydrolysis by MBLs, plus ceftazidime/avibactam (CZA) or meropenem/vaborbactam (M/V) for coverage of relevant SBLs. However, these selections add a level of complexity to clinical management compared with administration of a single antibiotic as monotherapy.MethodsMinimum inhibitory concentrations (MICs) of WCK 5222 (cefepime/zidebactam), ATM, CZA, and M/V were determined with Liofilchem MIC Test Strips against SBL- and MBL-positive CRE (N = 15). The gradient diffusion strip (GDS) cross method was used to assess the activities of CZA+ATM and M/V+ATM. Additive interactions as defined by fractional inhibitory concentration indices ≤ 1 would be predicted based upon the known genotypic profiles; thus, the relative activities of the combination regimens were compared with the “zone of hope” (ZOH) test. The size of the ZOH (the zone of inhibited growth) was quantitated by multiplying the observed length of inhibited growth (in mm) adjacent to each GDS from the point of intersection. The Mann–Whitney rank-sum test was used to assess differences.ResultsAll isolates (N = 15) contained one MBL and ≥1 SBL, and were resistant to ATM, CZA, and M/V with the exception of one isolate intermediate to M/V (MIC=8 mg/L). The WCK 5222 MIC50 (range) was 1 (0.19–2) mg/L. The median (interquartile range) ZOH product for CZA+ATM and M/V+ATM was 75.4 (62.8–93.7) and 23.5 (14.1–60.4), respectively (P = 0.002). In strains that produced OXA-type carbapenemases (n = 6), the median ZOH product for CZA+ATM and M/V+ATM was 78.1 and 20.7, respectively (P = 0.004). In the remaining 9 strains with a single carbapenemase (i.e., the MBL),the median ZOH product for CZA+ATM and M/V+ATM was 73.8 and 25.6, respectively (P = 0.052).ConclusionWCK 5222 displayed potent in vitro activity against SBL- and MBL-positive CRE, warranting further pre-clinical in vivo evaluation as a monotherapy option. When considering the co-expression of SBL and MBL, CZA+ATM appears to offer enhanced coverage compared with M/V+ATM.Disclosures All authors: No reported disclosures.

719. Cefiderocol Retains Anti-Biofilm Activity in MDR Gram-Negative Pathogens

BackgroundCefiderocol is a siderophore cephalosporin with potent antibacterial activity against a broad range of Gram-negative pathogens. Microorganisms forming biofilm, e.g., cUTI, utilize bacterial siderophores to access free iron. A siderophore antibiotic may have unique antimicrobial properties in the setting of biofilm. In this study, we compared antimicrobial activity of cefiderocol to comparator antibiotics in well-characterized multi-drug-resistant pathogens. We determined the activity of cefiderocol and comparator antibiotics in the biofilm setting.MethodsMinimum inhibitory concentrations (MICs) in Mueller–Hinton II broth (MHII) and iron-depleted cation-adjusted MHII (ID-CAMHB) were determined for cefiderocol and seven comparator antibiotics in multidrug-resistant isolates of P. aeruginosa, Burkholderia cepacia complex (Bcc), Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii. MBEC (minimum biofilm eradication concentration) assays were used to test cefiderocol’s activity in biofilms formed on pegs. Total biofilm biomass and viable cell number were measured.ResultsThe MIC90 of cefiderocol ranged from 0.125 μg/mL (Bcc) to 1 μg/mL (P. aeruginosa) in ID-CAMHB. MIC90 values were consistently lower for cefiderocol in all strains tested compared with other agents (ceftolozane-tazobactam, ceftazidime–avibactam, ceftazidime, pipercallin-tazobactam, imipenem, tobramycin, clarithromycin). Twenty-four hour P. aeruginosa biofilms (strains ATCC 9027, MB640, MB771, MB580A, MB730) were treated every 12 hours with 4 μg/mL of cefiderocol or comparator antibiotics. Cefiderocol treatment displayed a superior reduction in biofilm based on colony counts ( >90%; P < 0.0001 vs. untreated control) compared with comparator drugs (50 to 80% reduction). Crystal violet staining revealed a dose-dependent response of cefiderocol in the reduction of biofilm. Reduction of biofilm was not significantly altered by the growth media that was used; however, P. aeruginosa strains form more biofilm in MHII.ConclusionCefiderocol effectively reduces biofilm in multidrug-resistant strains of P. aeruginosa and is a potent inhibitor of planktonic growth across a range of Gram-negative medically important pathogens.Disclosures All authors: No reported disclosures.

Antimicrobial susceptibilities of the ertapenem-non-susceptible non-carbapenemase-producing Enterobacterales isolates causing intra-abdominal infections in the Asia-Pacific region during 2008–2014: Results from the Study for Monitoring the Antimicrobial Resistance Trends (SMART)

ObjectivesTo investigate the susceptibility profiles amongst ertapenem-non-susceptible non-carbapenemase-producing Enterobacterales (ETP-NS-non-CPE) isolates. MethodsMinimum inhibitory concentrations (MICs) of 404 ETP-NS-non-CPE isolates collected from different intra-abdominal infection (IAI) sites amongst patients in the Asia-Pacific region during 2008–2014 were determined using the broth microdilution method. The susceptibility results were interpreted according to the MIC breakpoints recommended by the Clinical and Laboratory Standards Institute (CLSI) in 2018. The MICs data of several agents were evaluated based on their published pharmacokinetic/pharmacodynamic (PK/PD) profiles. ResultsThe majority (>84%) of IAI-ETP-NS-non-CPE isolates – including Escherichia coli (n=83), Klebsiella pneumoniae (n=91) and Enterobacter species (n=210) – were susceptible to imipenem and amikacin. The 193 hepatobiliary ETP-NS-non-CPE isolates exhibited a trend of lower cefepime MIC (≤4mg/L) distribution than those (n=145) cultured from the peritoneal space (P=0.058). Amongst the ETP-NS-non-CP Enterobacter isolates, 65.7% displayed a cefepime MIC≤4mg/L. In addition, compared with Escherichia coli and Klebsiella pneumoniae isolates, 82.9% and 72.9% of the ETP-NS-non-CP Enterobacter isolates were susceptible to levofloxacin and ciprofloxacin, respectively. Of note, 74.5% and 70.3% of the ETP-NS-non-CP Enterobacter isolates cultured from the hepatobiliary tract and peritoneal space exhibited a ciprofloxacin MIC≤2mg/L and ≤0.25mg/L, respectively. Imipenem and amikacin showed good in vitro susceptibility rates against the IAI-ETP-NS-non-CPE isolates. The hepatobiliary ETP-NS-non-CPE displayed lower cefepime MICs than those cultured from the peritoneal space. Additionally, a significant fraction of IAI-ETP-NS-non-CP Enterobacter isolates exhibited ciprofloxacin MIC ≤ 2mg/L. ConclusionBased upon the PK/PD analyses, ciprofloxacin, imipenem and cefepime are probably effective against IAI-ETP-NS-non-CPE isolates.