MIC Results Research Articles

Identification of Antimicrobial Resistance-Associated Genes through Whole Genome Sequencing of Mycoplasma bovis Isolates with Different Antimicrobial Resistances.

Antimicrobial resistance (AMR) in Mycoplasma bovis has been previously associated with topoisomerase and ribosomal gene mutations rather than specific resistance-conferring genes. Using whole genome sequencing (WGS) to identify potential new AMR mechanisms for M. bovis, it was found that a 2019 clinical isolate with high MIC (2019-043682) for fluoroquinolones, macrolides, lincosamides, pleuromutilins and tetracyclines had a new core genome multilocus sequencing (cgMLST) type (ST10-like) and 91% sequence similarity to the published genome of M. bovis PG45. Closely related to PG45, a 1982 isolate (1982-M6152) shared the same cgMLST type (ST17), 97.2% sequence similarity and low MIC results. Known and potential AMR- associated genetic events were identified through multiple sequence alignment of the three genomes. Isolate 2019-043682 had 507 genes with non-synonymous mutations (NSMs) and 67 genes disrupted. Isolate 1982-M6152 had 81 NSMs and 20 disruptions. Using functional roles and known mechanisms of antimicrobials, a 55 gene subset was assessed for AMR potential. Seventeen were previously identified from other bacteria as sites of AMR mutation, 38 shared similar functions to them, and 11 contained gene-disrupting mutations. This study indicated that M. bovis may obtain high AMR characteristics by mutating or disrupting other functional genes, in addition to topoisomerases and ribosomal genes.

Susceptibility trends of ceftolozane/tazobactam and comparators when tested against European Gram-negative bacterial surveillance isolates collected during 2012-18.

The Program to Assess Ceftolozane/Tazobactam Susceptibility (PACTS) monitors the in vitro activity of ceftolozane/tazobactam and numerous antimicrobial agents against Gram-negative bacteria worldwide. To evaluate the activity of ceftolozane/tazobactam and resistance trends among Pseudomonas aeruginosa and Enterobacterales isolates in Europe between 2012 and 2018. P. aeruginosa (7503) and Enterobacterales (30 582) isolates were collected from 53 medical centres in 26 countries in Europe and the Mediterranean region and tested for susceptibility by reference broth microdilution method in a central laboratory. MIC results were interpreted using EUCAST criteria. Ceftolozane/tazobactam was the most active compound tested against P. aeruginosa isolates after colistin, with overall susceptibility rates of 94.1% in Western Europe and 80.9% in Eastern Europe. Moreover, ceftolozane/tazobactam retained activity against 75.2% and 59.2% of meropenem-non-susceptible P. aeruginosa isolates in Western and Eastern Europe, respectively. Tobramycin was the third most active compound tested against P. aeruginosa, with susceptibility rates of 88.6% and 70.9% in Western and Eastern Europe, respectively. Ceftolozane/tazobactam was active against 94.5% of all Enterobacterales and 96.1% of meropenem-susceptible isolates from Western Europe. In Eastern Europe, ceftolozane/tazobactam was active against 79.4% of Enterobacterales overall and 86.2% of meropenem-susceptible isolates. Antimicrobial susceptibility rates for agents commonly used to treat serious systemic infections varied widely among nations and geographic regions and were generally lower in Eastern Europe compared with Western Europe. Ceftolozane/tazobactam demonstrated potent activity against P. aeruginosa, including MDR strains, and retained activity against most meropenem-susceptible Enterobacterales causing infection in European medical centres.

Comparison of gentamicin MICs by agar dilution and Etest for clinical isolates of Neisseria gonorrhoeae.

In South Africa, Neisseria gonorrhoeae (NG) is the predominant cause of male urethritis syndrome (MUS). The national MUS treatment guidelines recommend gentamicin as salvage therapy for ceftriaxone treatment failures. We ascertained and compared gentamicin MICs obtained by agar dilution and Etest for clinical isolates of NG. Gentamicin MICs for NG culture isolates obtained from 272 MUS cases in 2017 were determined using agar dilution, as per CLSI agar dilution methods, and Etest® (bioMérieux, Marcy-l'Étoile, France). Previously published interpretive criteria were used: MIC ≤4 mg/L, susceptible (S); MIC 8-16 mg/L, intermediately resistant (IR); and MIC ≥32 mg/L, resistant (R). WHO 2008 NG reference strains were used as comparison standards. Gentamicin agar dilution versus Etest MIC results (mg/L) were as follows: MIC50 = 16 versus 4; MIC90 = 16 versus 8; minimum MIC = 4 versus 1; and maximum MIC = 32 versus 16. Interpretive categories for agar dilution versus Etest were as follows: S, 4.4% versus 86.8%; IR, 86.0% versus 13.4%; and R, 9.6% versus 0%. The gentamicin MIC50 by agar dilution was significantly higher than by Etest (sign test P value <0.001); overall MIC agreement was 7.4% [kappa statistic (κ) = -0.014 (95% CI -0.039 to 0.010)]. Correlation with expected MICs for WHO reference strains was consistently better with Etest than with agar dilution. There was a significant discordance between NG gentamicin MICs by agar dilution versus Etest. NG gentamicin AST methodology must be standardized and interpretive criteria established to optimize the monitoring of susceptibility trends.

Synthesis, antibacterial evaluation, in silico ADMET and molecular docking studies of new N-acylhydrazone derivatives from acridone

Novel N-acylhydrazone derivatives from acridone have been synthesized by condensation of acridone acetohydrazide and various aldehyde. The novel acylhydrazones were tested for their in-vitro antibacterial activity against human pathogenic strains. The MIC results indicate that compound 3f displayed high antibacterial potential against Pseudomonas putida with MIC = 38.46 µg/mL, which is very close to that obtained with the commercial antibiotic. The synthesized compounds were subjected for docking studies to understand the interaction of our compounds and transcriptional regulator enzyme of pseudomonas putida and DNA gyrase complex of Staphyloccocus aureus.

UJI AKTIVITAS ANTIBAKTERI SEDIAAN OBAT KUMUR EKSTRAK ETANOL DAUN MANGGA HARUMANIS (Mangifera indica, L) TERHADAP STREPTOCOCCUS MUTANS PENYEBAB KARIES GIGI

Dental caires is a disease of the oral cavity caused by the bacterium Streptococcus mutants. Treatment is usually done by using mouthwash which if used continuously will cause side effects. Therefore, need to develop mouthwash preparations from natural ingredients. One of the potential plants is mango harumnais (Mangifera Indica, L) leaves which contain flavonoids, saponins, tannins and polyphenols as antibacterial. This study aims to determine the activity of the ethanol extract of haruman mango leaves mouthwash that can affect the growth of streptococcus mutants’ bacteria and produce stable preparations during the storage period. By varying the concentration of harumanis mango leaf extracts of 10, 15, and 20%, the concentration was based on the MIC results of the ethanol extract of harumanis mango leaf. The results of testing preparations that have the potential as an antibacterial are in formula III with the formation of the largest inhibitory zone of 5.35 mm, formula II 5.06 mm, formula III 3.26 mm. The mouthwash of ethanol extract of harumanis mango leaves (Mangifera Indica, L) meets the requirements and the preparation is stable in a 30-day storage period.

Screening and Estimation of Secondary Metabolites in Leaf Extracts of Delonix elata (L.) Gamble and their Inhibitory Activities against Pathogens and Oxidative Stress

Delonix elata (L.) Gamble is a tree commonly available and well known to be used for joint pain, flatulence and arthritic disorders. The main objective of this study was to investigate biological activities of the phytochemicals of leaf extracts of Delonix elata (L.) for activities against pathogens and oxidative stress. The plant source was sequentially extracted by the following solvents chloroform, ethyl acetate, acetone, methanol and water. Antibacterial activity was tested against six pathogens with disc diffusion method; MIC and MBC&nbsp;were also done by double dilution method. The standard procedures were followed for estimation of flavonoids, flavanones, phenolic compounds and alkaloids. Antioxidant potential was determined by DPPH assay. Obtained five different solvent extracts and observed clear zone of inhibitions against all the pathogens on the extract of ethyl acetate. The MIC and MBC results were observed against all the pathogens, compared to other pathogens Staphyl-ococcus aureus inhibits at very low concentration (30mg/mL) of the extract. The rich source of flavonoids and phenolic compounds present in aqueous extract, flavanones present in methanol extract and alkaloids present in ethyl acetate extract. As the antioxidant assay, higher percentage (82.82%) of inhibition was calculated in ethyl acetate extract. On phytochemical screening the presence of phenols, flavonoids, alkaloids, steroids, terpenoids, carbohydrates, proteins and aminoacids were observed. This investigation shows the potential inhibitory activity of ethyl acetate extract of the plant leaves. The phytoconstituents are rich sources present in the different solvent extracts of D. elata leaf that may be responsible for the potential antimicrobial and antioxidant activity. S. aureus is one of the leading causes of infection in hospital. Here we found that D. elata leaf extract inhibit the pathogen S. aureus at very low concentration (30mg/mL) itself. Therefore, it is suggested that these leaves are helpful for identification of new drugs against this pathogen.

Preparation of Barije (Ferula gummosa) Essential Oil–Loaded Liposomes and Evaluation of Physical and Antibacterial Effect on Escherichia coli O157:H7

The aim of this study was to load liposomes with Barije (Ferula gummosa) essential oil (EO) and to evaluate their physical and antibacterial properties. Liposomes were produced with specific ratios of lecithin/cholesterol by thin-film hydration and sonication. The chemical composition of the EO was analyzed by gas chromatography and mass spectroscopy. The physical properties of the liposomes (particle size, polydispersity index, zeta potential, and encapsulation efficiency) were evaluated. The antimicrobial effects of these liposomes against Escherichia coli O157:H7 were determined based on the MIC and disk diffusion results. The effect of subinhibitory concentrations (sub-MICs) of EO against the growth of the bacterium over 24 h was evaluated before and after encapsulation. The major components of EO were β-pinene (60.84%) and α-pinene (9.14%). The mean liposome radius of EO-loaded liposomes was 74.27 to 99.93 nm, which was significantly different from that of the empty liposomes (138.76 nm) (P < 0.05). Addition of cholesterol to the lecithin bilayer increased the particle size and reduced the encapsulation efficiency (P < 0.05). The electrostatic stability of the empty liposomes was improved by adding cholesterol, but when the EO was replaced in the liposomes, there was no significant change in electrostatic stability of liposomes with cholesterol (P < 0.05). MICs were 14.5 μg/mL for the EO-loaded nanoliposomes containing 30 mg of lecithin and 30 mg of cholesterol and 10 μg/mL for nonencapsulated EO. This trend was confirmed by measuring the inhibition zone diameter. Sub-MICs of liposomal EO (containing 60 mg of lecithin) decreased bacterial levels to a greater degree than did free EO, especially at 50 and 75% of the MIC.

Evaluation of EUCAST rapid antimicrobial susceptibility testing (RAST) for positive blood cultures in clinical practice using a total lab automation

Our objective was to evaluate EUCAST’s ‘rapid antimicrobial susceptibility testing’ (RAST) directly from positive blood culture that delivers antimicrobial results within 6 h for Staphylococcus aureus, Enterococcus spp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, using total lab automation. Zone diameters from RAST were compared with MIC results. Furthermore, its influence on time to report was investigated. RAST was performed to all positive aerobic and anaerobic blood culture bottles by subculturing them, i.e. onto Mueller-Hinton agar and adding six antibiotic discs covering Gram-negative and Gram-positive therapy (cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin). RAST was automatically imaged after 6 h. Zone sizes were measured using a TLA software tool and interpreted according to EUCAST clinical breakpoints. Bacteria were identified using MALDI-TOF MS and MIC results were determined using Vitek2 panels. Categorial agreement between agar diffusion and MIC results was investigated. Additionally, time to RAST and time to Vitek were compared for 100 isolates (20 per species). Between November 2018 and April 2019, 3313 positive mono-bacterial blood culture bottles were collected of which 894 bottles with RAST-validated species were investigated. Among these bottles, 2029 individual antibiotic measurements were compared with MIC results from Vitek2 and 14 very major, 28 major and 12 minor errors were found. A median reduction of 17:30 h in time to report was observed. Introduction of RAST with automatic TLA imaging function could reduce time to report by 17:30 h. Excellent accordance between zone diameter and MIC results, particularly for cefoxitin, vancomycin and meropenem, was observed, but drawbacks due to ATU were seen.

NEW 2,4,5-TRISUBSTITUTED-1,3-THIAZOLE DERIVATIVES: SYNTHESIS, IN VITRO ANTIMICROBIAL ACTIVITY AND IN SILICO STUDY

A series of thiazole derivatives 1-4 were synthesized employing simple one-pot reaction pathway and characterized via Fourier Transform Infrared (FTIR), Proton Nuclear Magnetic Resonance (1H NMR), Ultraviolet-Visible (UV-Vis) and Gas Chromatography-Mass Spectrometry (GC-MS). The newly synthesized compounds were evaluated for their in vitro antimicrobial properties against several bacterial strains including Gram-positive and Gram-negative as well as fungus using broth microdilution method. The results revealed that all of the compounds exhibited good activity with a range of MIC values between 1.25-5.0 mg/mL. From the MIC and MBC results, compound 1 exhibited good activities with same MIC value of 1.25 mg/mL and MBC value of 5 mg/mL against B. cereus and S. flexneri. In order to support antimicrobial results, the molecular docking studies were carried out for inhibition of the GlcN-6-P synthase as the target. Out of four compounds underwent for molecular docking studies, 5-acetyl-4-methyl-2-(4-aminobiphenyl)-1,3-thiazole (1) shows the lowest minimum binding energy at -7.32 kcal/mol as compared to 2, 3 and 4 with -7.31, -7.20 and -6.76 kcal/mol, respectively which are in agreement with antimicrobial assay results. In conclusion, 2, 4, 5-trisubstituted- 1,3- thiazole derivatives could be considered as promising antimicrobial in drug discovery candidates.

Design, Synthesis, Docking and Biological Evaluation of Novel 4-hydroxy Coumarin Derivatives.

Hospital-acquired (HA) infections are caused due to E. coli, which is resistant to multiple drugs particularly to fluoroquinolone class of drugs. Urinary tract infections (UTI) affects people in the community and hospitals. 150 million people per annum are suffering from UTI worldwide. In this present study, we designed 36 novel coumarin derivatives, also we predicted pharmacokinetic and toxicity parameters. Docking studies were also carried out and all the compounds were evaluated for antibacterial activity against resistant quinolone E. coli strain ATCC 25922. It was interesting to note that the introduction of electron-withdrawing group on the aromatic ring resulted in compounds with an increased antibacterial activity, which is observed in compound 6 (with 4-nitro substitution), compound 23 (chloro) and compound 30 (chloro, nitro). From the MIC results, it was observed that compounds 6, 23 and 30 showed higher activity with 0.5μg/ml, 0. 12 μg/ml, 0.5 μg/ml respectively. Docking studies were performed with the active site of DNA gyrase (PDB ID: 4CKK). The maximum binding energy was found to be -10.7 Kcal/mol. From the study, it was found that 3 compounds were potentially active against quinolone- resistant E. coli strains. This study can further be extended for in vivo evaluation.

DETECTION OF ANTIBIOTICS RESISTANCE GENES IN STAPHYLOCOCCUS AUREUS ISOLATED FROM POULTRY FARMS

Poultry industry in Egypt was subjected to various problems one of them was the early chick mortalities that cause great economic losses and its investigation is the request of many poultry scientists and men. In poultry staphylococci, including Staphylococcus aureus are known to cause various diseases from acute septicemia to chronic osteomyelitis. Two hundred and sixty samples of chickens, ducks and turkeys were collected from different poultry farms. A survey on Staphylococcal infection among chickens, ducks and turkeys was carried out. It was found that out of 260 infected and dead chickens, ducks and turkeys suspected to be infected with Staphylococcosis, 54 cases (20.76%) revealed Staphylococcus micro-organisms. The isolated strains were typed as: Staphylococcus aureus (74.07%), Staphylococcus epidermidis (14.8 %)and Staphylococcus saprophyticus (11%). The results of sensitivity biogram revealed that S. aureus were highly sensitive to Amoxicillin, Ampicillin, Ciprofloxacine, Enrofloxacine and lincomycin and resistant to Erythromycin, Streptomycine and Chloramphenicol. The results of MIC of 6 representative coagulase positive Staphylococci isolates against 6 selected antibiotics commonly used in poultry farms showed that 100% of isolates were sensitive to amoxicillin, enrofloxacin, ciprofloxacin and oxytetracyclin, while 100% of 6 isolates were resistant to streptomycin and lincomycin. The incidence of isolation of Staphylococci from the internal organs of examined birds and from unabsorbed yolk sac, joints, liver, intestinal content and heart blood was 36.3%, 20%, 19.2%, 17.3% and 16.9% respectively. Ten of each detected isolates were examined by cPCR for resistance genes blaZ and aac(6’) aph (2’’). The isolates harbored these resistance genes with percentage of 100% for S. aureus. In experiment, the pathogenicity of the isolated strains of S. aureus for 7 day- old chicks was studied. S. aureus injected subcutaneously, oral and intranasal caused death of 100%, 100% and 26.7% of the used chicks respectively.

1583. Eight Years of Sustained Potency and Activity of Oritavancin against Gram-Positive Isolates Causing Bacteremia and Endocarditis in the USA, Including Enterococcal Infections Requiring an Optimized Dosing Strategy for Daptomycin

BackgroundOritavancin (ORI) is a potent lipoglycopeptide with desirable PK/PD parameters for treating serious gram-positive infections. This study assessed the activity of ORI against Staphylococcus aureus (SA), Enterococcus faecalis (EF), and E. faecium (EFM) causing bloodstream infection (BSI), including infective endocarditis (IE) and daptomycin (DAP)- susceptible dose-dependent (SDD) vancomycin-resistant (VRE) subsets. We also evaluated the longitudinal activity of ORI.MethodsA total of 5,469 SA, 1,157 EF, and 721 EFM were recovered from BSI in 35 US sites (2011–2018). Subsets of SA isolates causing IE (84) and EFM displaying DAP-SDD-VRE phenotypes (230) were included. Identification was confirmed by MALDI-TOF MS and isolates were tested for susceptibility (S) according to CLSI.ResultsOverall, ORI showed similar MIC50 (0.03 mg/L) and MIC90 results (0.06 mg/L) against MRSA and MSSA (figure) and the SA EC subset (41.7% MRSA; data not shown). Similar findings were noted for ORI tested against EF DAP-S (MIC50/90, 0.015/0.06 mg/L) and DAP-SDD (MIC50/90, 0.015/0.06 mg/L). ORI MIC values against DAP- and VAN-S EFM (MIC50/90, ≤0.008/0.015 mg/L) were at least 8-fold lower than those from DAP-SDD-VRE isolates (MIC50/90, 0.06/0.12 mg/L; 31.9% of all EFM), and all EFM were inhibited by ORI at ≤0.25 mg/L. The longitudinal analysis showed MRSA rates varying from 39.7% (2017) to 46.8% (2011), while the annual ORI MIC50 and MIC90 results were 0.015–0.06 mg/L and 0.03–0.12 mg/L, respectively, against MRSA during the 8-year period. ORI yearly MIC50 and MIC90 results were 0.015–0.03 mg/L and 0.03–0.12 mg/L against EF, respectively. MIC50 and MIC90 results of 0.008–0.03 mg/L and 0.03–0.12 mg/L, respectively, were obtained for ORI against the DAP-SDD EF subset each year. ORI MIC50 and MIC90 results of 0.03–0.06 and 0.06–0.12 mg/L were obtained annually against DAP-SDD-VRE (EFM), respectively.ConclusionORI showed a potent activity against this collection of isolates causing BSI and IE in the USA, including resistant subsets requiring higher dosage regimens when treating serious infections. In addition, ORI maintained a stable potency throughout the 8-year study period with no apparent temporal trends. Disclosures All authors: No reported disclosures.

2132. Multicenter Evaluation of Eravacycline MIC Results for Enterobacteriaceae Using MicroScan Dried Gram-Negative MIC Panels

BackgroundA multicenter study was performed to evaluate the accuracy of eravacycline on a MicroScan Dried Gram-negative MIC (MSDGN) Panel when compared with a frozen CLSI broth microdilution reference panel.MethodsFor efficacy, an evaluation was conducted at three sites by comparing MIC values obtained using the MSDGN to MICs using a CLSI broth microdilution reference panel. A total of 414 Enterobacteriaceae clinical isolates were tested using the turbidity and Prompt®* methods of inoculation. For challenge, 79 Enterobacteriaceae isolates were tested on MSDGN panels at one site. For reproducibility, a subset of 11 organisms was tested on MSDGN panels at each site. MSDGN panels were incubated at 35 ± 2°C and read on the WalkAway System, the autoSCAN-4 instrument, and read visually. Read times for the MSDGN panels were at 16–20 hours. Frozen reference panels, prepared according to CLSI/ISO methodology, were inoculated using the turbidity inoculation method. All frozen reference panels were incubated at 35 ± 2°C and read visually. Frozen reference panels were read at 16–20 hours. FDA breakpoints (µg/mL) used for interpretation of MIC results were: Enterobacteriaceae ≤ 0.5 S. Potential major and very major errors were calculated using the NS result in place of resistant (R).ResultsWhen compared with frozen reference panel results, essential and categorical agreements for isolates tested in the Efficacy and Challenge are as follows (see table). Reproducibility among the three sites were greater than 95% for all read methods for both the turbidity and Prompt inoculation methods.ConclusionThis multicenter study showed that eravacycline MIC results for Enterobacteriaceae obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels using FDA interpretive criteria. * PROMPT® is a registered trademark of 3M Company, St. Paul, MN USA. Beckman Coulter, the stylized logo and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. Xerava™ (Eravacycline) is a registered trademark of Tetraphase Pharmaceuticals, Inc. Disclosures All authors: No reported disclosures.

2131. Multicenter Evaluation of Meropenem/Vaborbactam MIC Results for Enterobacteriaceae Using MicroScan Dried Gram-Negative MIC Panels

BackgroundA multicenter study was performed to evaluate the accuracy of meropenem/vaborbactam on a MicroScan Dried Gram-negative MIC (MSDGN) Panel when compared with a frozen CLSI broth microdilution reference panel.MethodsFor efficacy, an evaluation was conducted at three US sites by comparing MIC values obtained using the MSDGN to MICs using a CLSI broth microdilution reference panel. A total of 560 Enterobacteriaceae clinical isolates were tested using the turbidity and Prompt®* methods of inoculation. For challenge, 95 Enterobacteriaceae isolates were tested on MSDGN panels at one site. For reproducibility, a subset of 14 organisms was tested on MSDGN panels at each site. MSDGN panels were incubated at 35 ± 2°C and read on the WalkAway System, the autoSCAN-4 instrument, and read visually. Read times for the MSDGN panels were at 16–20 hours. Frozen reference panels, prepared according to CLSI/ISO methodology, were inoculated using the turbidity inoculation method. All frozen reference panels were incubated at 35 ± 2°C and read visually. Frozen reference panels were read at 16–20 hours. FDA/CLSI breakpoints (µg/ml) used for interpretation of MIC results were: Enterobacteriaceae ≤ 4/8 S, 8/8 I, and ≥ 16/8 R.ResultsWhen compared with frozen reference panel results, essential and categorical agreements for isolates tested in the Efficacy and Challenge are as follows (see table). Reproducibility among the three sites were greater than 95% for all read methods for both the turbidity and Prompt* inoculation methods.ConclusionThis multicenter study showed that meropenem/vaborbactam MIC results for Enterobacteriaceae obtained with the MSDGN panel correlate well with MICs obtained using frozen reference panels using FDA/CLSI interpretive criteria. * PROMPT® is a registered trademark of 3M Company, St. Paul, MN, USA. Beckman Coulter, the stylized logo and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. Vabomere® (Meropenem/Vaborbactam) is a registered trademark of Melinta Therapeutics, Inc. Disclosures All authors: No reported disclosures.

715. Bulgecin A lowers Imipenem MICs in Clinical MDR Acinetobacter baumannii (ACB) Strains

BackgroundGenetic screening of ACB has revealed genes that confer increased susceptibility to β-lactams when these genes are disrupted suggesting novel drug targets. One such target is lytic transglycosylase, LT. Bulgecin A (BlgA) is a natural product of Paraburkholderia acidophila and an LT inhibitor that potentiates the activity of β-lactams.MethodsBroth microdilution MICs were performed using carbapenem-resistant (CR)-colisitin susceptible (ColS) and CR-ColR clinical ACB strains, with a variety of resistance mechanisms, previously studied via whole-genome sequencing. A fixed concentration of pure BlgA at 100 μg/mL was combined with varying concentrations of imipenem. Sequences of the putative LTs in ACB strains were analyzed to look for amino-acid substitutions and correlated with the MIC lowering effect of BlgA. Homology models of the LTs of AB0057 ACB strain were generated.ResultsACB MltE most resembles soluble LTs of other species with 22.39% sequence identity and 92% query coverage to Slt70 of E. coli. MIC results and amino-acid sequence variations in MltE LT of ACB are shown. There were no clear amino-acid substitution patterns to account for differences in BlgA effect. The mutations are distal to the BlgA binding site at residue E506.ConclusionThese are the first MICs reported for BlgA in combination with a carbapenem demonstrating reduction in the MIC by four-fold for several MDR ACB clinical strains. Mutations in MltE Lt do not account for variations in the MICs observed. Changes in the MICs may be related to other factors such as drug penetration and alterations in PBP expression. Additional studies are underway to examine these factors. Disclosures All authors: No reported disclosures.

714. Analysis of the Effect of Urine on the In Vitro Activity of Gepotidacin and Levofloxacin Against Escherichia coli, Staphylococcus epidermidis, and Staphylococcus saprophyticus

BackgroundGepotidacin (GSK2140944) is a first in class novel triazaacenaphthylene bacterial type II topoisomerase inhibitor in clinical development for the treatment of gonorrhea and uncomplicated UTI (acute cystitis). Gepotidacin selectively inhibits bacterial DNA gyrase and topoisomerase IV by a unique mechanism not utilized by any currently approved therapeutic agent and demonstrates in vitro activity against most target pathogens resistant to established antibacterials, including fluoroquinolones. This study was undertaken to determine the effect of various urine parameters on the in vitro activity of gepotidacin and a comparative agent, levofloxacin, against a variety of bacteria.MethodsStudy strains were tested according to the reference CLSI broth microdilution method using cation-adjusted Mueller–Hinton broth (CAMHB) and the following method variations: CAMHB with 25%, 50%, and 100% urine (not pH adjusted) and 100% urine (pH adjusted to 7.2–7.4, and 8). Quality control strains were concurrently tested each day.ResultsMIC endpoints in the reference method and in 100% urine were easily determined (i.e., clear buttons of growth up to the first well of no growth). Gepotidacin MIC results are summarized in the table. For E. coli and S. saprophyticus, there was a trend for higher gepotidacin MIC results with the addition of increasing amounts of urine. However, the increase was minimal such that mean dilution differences were ≤ 1.54. Against S. epidermidis, gepotidacin MICs were not significantly impacted by the addition of urine as 100% of urine condition MICs were within ±1 doubling dilution of the reference method MIC. The gepotidacin results for E. coli indicate that the average 1–2 dilution MIC increase observed in the unadjusted 100% pooled urine MIC may be associated with lower pH. A similar increase in levofloxacin MIC results for E. coli were also associated with pH, but at the higher pH of 8.0. In contrast, the increase in MIC observed in both the gepotidacin and levofloxacin S. saprophyticus results in 100% pooled urine do not appear to be a function of pH.ConclusionOverall, the effect of urine on the gepotidacin and levofloxacin MICs was minimal and not inclusive of all strains tested. Disclosures All authors: No reported disclosures.

Development of bioconjugated nano-molecules against targeted microbial pathogens for enhanced bactericidal activity

The study reports development of bio-conjugated nano-molecules (BCM's) for enhanced antibacterial activity against targeted pathogens of global concern. The conjugation was achieved with biomolecular interaction of silver clusters with 3-Dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) which was purified and characterized with TLC which displayed prominent band at Rf 0.5 and HPLC analysis displayed chromatographic peak eluting at (tR) 6–8 min. The LC-MS analysis displayed single major peak at tR = 4.610 corresponding to molecular ion peak at m/z = 227.19. The developed BCM molecule was determined with UV–Visible spectroscopy which displayed the absorbance peaks of conjugated molecules with shoulder peak observed and morphological characteristics were well defined with TEM analysis which showed cluster formation. The size ranged from 20 to 80 nm in size with majority of the BCM displaying spherical in size. The process of bio-conjugation was further studied with FTIR analysis which corresponded with different vibrational stretches owing to the presence of amide, carbonyl alkynes, nitriles and carboxylic acid groups. The presence of functional moieties was also studied using 1H and 13C NMR spectra. The crystalline characteristics was confirmed with XRD analysis which displayed Bragg's intensities along with additional peaks occurring at 2 theta angle corresponds to (111), (200), (220), (311) face centric cube of silver planes. The antibacterial activity of BCM's was profound against all the test pathogen which was validated and compared with standard antibiotics. Among the test pathogens, highest activity was conferred against Staphylococcus epidermidis (MTCC 435) with 29 mm. The minimal inhibition concentration of BCM's was in the range of 0.97–3.12 μg/ml. The results of MIC were in accordance with well diffusion assay indicating the Gram + ve test pathogen Staphylococcus epidermidis to be the most sensitive. In addition, the broth dilution assay resulted in decrease in the optical density measured at 600 nm against the increase in the concentration of BCM's. The outcome of the present investigation revealed the role of bio-conjugation chemistry to increase fold activity against pathogens which can act as alternative tool to combat drug resistant menace across the globe.

Point-Counterpoint: Differences between the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute Recommendations for Reporting Antimicrobial Susceptibility Results

INTRODUCTIONAntibiotic susceptibility test results are among the most important results issued by clinical microbiology laboratories because they routinely guide critical treatment decisions. Interpretations of MIC or disk diffusion test results, such as "susceptible" or "resistant," are easily understood. Clinical laboratories also need to determine whether and how their reports will reflect more complex situations. Such situations include, first, whether there is need to administer higher or more frequent doses of antibiotic than usual for clinical efficacy; second, whether an antimicrobial is likely to be effective at a body site where it concentrates; and third, whether there is some uncertainty in the test results due to technical variability that cannot be eliminated. Two leading organizations that set standards for antimicrobial susceptibility testing, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI), have taken different strategies to deal with these challenges. In this Point-Counterpoint, Gunnar Kahlmeter and Christian Giske discuss how EUCAST is addressing these issues, and Thomas Kirn and Susan Sharp discuss the CLSI approach.

Peptide nucleic acid-mediated re-sensitization of colistin resistance Escherichia coli KP81 harboring mcr-1 plasmid

Escherichia coli is a gram-negative bacterium and it causes a variety of diseases in humans. It causes a wide range of clinical infections in humans; urinary tract infections is the most prevalent infection caused by uropathogenic Escherichia coli. In recent years, the observation of antibiotic-resistant genes such as resistance to colistin, makes the Escherichia coli resistant to antibiotics like colistin (polymyxin E), because of that the use of new therapies like peptide nucleic acid (PNA) has attracted the consideration of scientists. The aim of this study is the assessment of the inhibitory role of PNA against mcr-1 gene and reduction of mcr-1 gene expression and MIC in colistin resistant E. coli by PNA. NCBI database was used to design PNA. Our study was carried out on E. coli KP81 bacteria containing the mcr-1 gene. Microbroth dilution (MIC) method was used to survey phenotypic sensitivity and determine the sensitivity of the bacteria to the colistin antibiotic. E. coli KP81 isolates were further investigated by polymerase chain reaction to assess the presence of mcr-1 genes and target genes were quantified by real-time PCR assay using specific primers. The MIC result after treatment with specific PNA showed that the resistance to colistin reduced about three fold and the resistance level dropped from 32 μg/ml to 4 μg/ml. The expression analysis of mcr-1 gene in E. coli KP81 isolate indicates the PNA, 95% reduced the expression of the mcr-1 gene. Our observations showed that by inhibiting the expression of mcr-1, sensitivity to colistin can be defeated. Using higher concentrations of PNA and an in vivo study can reveal more clinical application of this method.

Pharmacological and Biological Evaluation of 5,5’[(1,4-Phenelene) bis (1,3,4-thiadiazol-2-amine)

The thiadiazole 2-amines compound have been synthesis in this study. The biological activity of the compound was determined at 100, 150, 200 and 250 mg/ml against four types of bacteria i.e. Bacillus cereus, Staphylococcus aureus, Escherichia coli and Slmonella sp. The results of MIC were 2,5,4,5 mg/ml for Bacillus cereus, Staphylococcus aureus, Escherichia coli and Slmonella spp, respectively. Median lethal dose (LD50) and cytotoxic activity were also investigated. The results showed that this compound did not affect the red blood until the concentration reach 30mg/ml or above. The LD50 for this compound is 0.01994 gm/kg bw.