In vitro Time Kill of Trimethoprim/Sulfamethoxazole against Stenotrophomonas maltophilia versus Escherichia coli using Cation Adjusted Muller Hinton Broth and ISO-Sensitest Broth

Trimethoprim/sulfamethoxazole (TMP/SMZ) is considered the treatment of choice for infections caused by Stenotrophomonas maltophilia , but limited pharmacodynamic data are available to support current susceptibility breakpoints or guide optimal dosing. Time-kill studies using a TMP/SMZ concentration of 4/40 μg/mL were conducted to compare 4 S. maltophilia with 4 Escherichia coli having the same MICs (0.25/4.75-4/76 μg/mL) in cation adjusted Mueller Hinton Broth (CAMHB) and ISO-Sensitest™ broth (ISO). With the exception of the resistant isolates (4/76 μg/mL), which resulted in regrowth approaching control, TMP/SMZ displayed significantly greater killing for E. coli compared with S. maltophilia at each MIC. Against E. coli , mean changes at 24 hour were -4.49, -1.73, -1.59, and +1.83 log 10 colony forming units (CFU) for isolates with MICs of 0.25/4.75, 1/19, 2/39, and 4/74 μg/mL, respectively. The f AUC/MIC required for stasis, 1-log 10 , and 2-log 10 CFU reductions were 40.7, 59.5, and 86.3, respectively. In contrast, TMP/SMZ displayed no stasis or CFU reductions against any S. maltophilia regardless of MIC, and no pharmacodynamic thresholds were quantifiable. Observations were consistent in both CAMHB and ISO broth. These data add increasing evidence that current TMP/SMZ susceptibility breakpoints against S. maltophilia should be reassessed.

1287. Double Disk Diffusion Study to Evaluate the Synergistic Effect Between Cefiderocol and Ceftazidime-Avibactam Against Cefiderocol-Non-susceptible Acinetobacter baumannii

Background Cefiderocol (CFDC), a siderophore cephalosporin, has broad coverage of Gram-negative bacteria and has been approved for clinical use in USA and Europe. The SIDERO-WT surveillance studies showed that CFDC shows >95% susceptibility against Acinetobacter baumannii. Against many of CFDC non-susceptible isolates, most of which had blaPER gene, the combination use of avibactam significantly decreased the MIC by broth microdilution (BMD). In this study, we evaluated the appropriate methodology to evaluate the synergistic effect by disk diffusion studies. Methods The susceptibility testing was conducted as recommended by the CLSI using CFDC non-susceptible isolates (MIC of >4 µg/mL based on CLSI breakpoint). The MIC by BMD was determined using iron-depleted cation-adjusted Mueller-Hinton broth, in the presence or absence of 4 µg/mL of avibactam. The disk diffusion was evaluated using Mueller-Hinton agar, and the synergy was evaluated by using disk stacking methods. For disk stacking methods, CFDC disk was placed on agar on which bacterial suspension of 0.5 McFarland units was spread, then the ceftazidime-avibactam (CZA) disks was stacked on the top, followed by adding a drop (30 µL) of saline on the stacked disks. As an alternative method, CZA was immersed in saline for 1 second instead of adding a drop of saline, followed by the stacking on the top. The disk zone size was determined after 24-hour incubation at 37°C. Results Against blaPER-positive A. baumannii which showed >64 µg/mL MIC of CFDC and CZA, CFDC MIC decreased to 0.25 µg/mL in the presence of avibactam. The disk diffusion methods also showed isolates resistant to CFDC and CZA and showed susceptiblilty disk zone to CFDC by stacking both disks. On the other hand, against blaNDM-positive A. baumannii which showed 64 µg/mL MIC of CFDC and CZA, the disk diffusion methods showed resistance even when stacking both disks. Against multiple isolates, the MIC of CFDC without or with avibactam was correlated well with the disk zone produced by CFDC disk alone or stacked with CZA disks, respectively (Figure). Conclusion The synergistic effect between CFDC and avibactam by BMD methods could be detected by disk stacking methodology using CFDC and CZA disks. Disclosures Yoshinori Yamano, PhD, Shionogi (Employee) Miki Takemura, MS, SHIONOGI & CO., LTD. (Employee) Roger Echols, MD, Shionogi (Consultant) Christopher Longshaw, PhD, Shionogi (Employee)

PK/PD Analysis by Nonlinear Mixed-Effects Modeling of a Marbofloxacin Dose Regimen for Treatment of Goat Mastitis Produced by Coagulase-Negative Staphylococci

Coagulase-negative staphylococci are main pathogens that produce goat mastitis. Marbofloxacin is a third-generation fluoroquinolone approved for treat mastitis in animals. The objectives of this study were: (i) to determine the pharmacokinetics of marbofloxacin (10 mg/kg/24 h) in serum and milk administered intramuscularly for five days in goats with mastitis induced by coagulase-negative staphylococci; (ii) to characterize the concentration–effect relationship of marbofloxacin against coagulase-negative staphylococci in Mueller Hinton broth and goat milk; (iii) to determine AUC/MIC cutoff values of marbofloxacin, and (iv) to perform a PK/PD analysis to evaluate the efficacy of the dose regimen for the treatment of goat mastitis produced by coagulase-negative staphylococci. Marbofloxacin presented context-sensitive pharmacokinetics, influenced by the evolution of the disease, which decreased marbofloxacin disposition in serum and milk. Marbofloxacin showed a median (95%CI) fAUC/MIC values for MIC of 0.4 and 0.8 µg/mL of 26.66 (22.26–36.64) and 32.28 (26.57–48.35) related with −2 log10CFU/mL reduction; and 32.26 (24.81–81.50) and 41.39 (29.38–128.01) for −3 log10CFU/mL reduction in Mueller Hinton broth. For milk, −2 log10CFU/mL reduction was achieved with 41.48 (35.29–58.73) and 51.91 (39.09–131.63), and −3 log10CFU/mL reduction with 51.04 (41.6–82.1) and 65.65 (46.68–210.16). The proposed dose regimen was adequate for the treatment of goat mastitis produced by coagulase-negative staphylococci, resulting in microbiological and clinical cure of all animals. The animal model used in this study provided important pharmacokinetic information about the effect of the infection on the pharmacokinetics of marbofloxacin. Pharmacodynamic modeling showed that fAUC/MIC cutoff values were higher in goat milk compared with Mueller Hinton broth.

Melanin: Production from Cheese Bacteria, Chemical Characterization, and Biological Activities

Pigments are compounds of importance to several industries, for instance, the food industry, where they can be used as additives, color intensifiers, and antioxidants. As the current trend around the world is shifting to the use of eco-friendly commodities, demand for natural dyes is increasing. Melanins are pigments that are produced by several microorganisms. Pseudomonas putida ESACB 191, isolated from goat cheese rind, was described as a brown pigment producer. This strain produces a brown pigment via the synthetic Müeller-Hinton Broth. This brown compound was extracted, purified, analyzed by FTIR and mass spectrometry, and identified as eumelanin. The maximum productivity was 1.57 mg/L/h. The bioactivity of eumelanin was evaluated as the capacity for scavenging free radicals (antioxidant activity), EC50 74.0 ± 0.2 μg/mL, and as an acetylcholinesterase inhibitor, with IC50 575 ± 4 μg/mL. This bacterial eumelanin did not show cytotoxicity towards A375, HeLa Kyoto, HepG2, or Caco2 cell lines. The effect of melanin on cholesterol absorption and drug interaction was evaluated in order to understand the interaction of melanin present in the cheese rind when ingested by consumers. However, it had no effect either on cholesterol absorption through an intestinal simulated barrier formed by the Caco2 cell line or with the drug ezetimibe.

Determination of Tedizolid in Bacterial Growth Medium Mueller-Hinton Broth by High-Performance Liquid Chromatography and Its Application to an In Vitro Study in the Hollow-Fiber Infection Model

Pharmacokinetic/pharmacodynamic (PKPD) studies of anti-infectives are frequently performed in in vitro infection models where accurate quantification of antibiotic concentrations in bacterial growth media is crucial to establish PK/PD relationships. Here, a sensitive and rapid high-performance liquid chromatography (HPLC) method was developed to quantify tedizolid (TDZ) in the bacterial growth medium Mueller-Hinton broth (MHB). Matrix components were separated by direct protein precipitation with methanol (1:1). The chromatographic separation was carried out in a Dionex Ultimate 3000 HPLC system using an Accucore® C-18 RPMS HPLC column (2.6 µm, 100 × 2.1 mm) using isocratic elution with 25% acetonitrile and 75% of 0.1% formic acid. The lower limit of quantification was 0.03 mg/L when measured at 300 nm. Following relevant European Medicine Agency guidelines, the method was successfully validated for linearity, selectivity, recovery, inter- and intra-day precision, and accuracy and stability. When applied to in vitro PKPD studies, the method successfully quantified a range of TDZ concentration (Cmin, 0.09-Cmax, 0.65 mg/L) in MHB. The analyzed concentrations were in line with the planned PK profiles. The application of the developed method to quantify TDZ in MHB in in vitro PKPD studies is warranted.

Recording the Presence of Peanibacillus larvae larvae Colonies on MYPGP Substrates Using a Multi-Sensor Array Based on Solid-State Gas Sensors

American foulbrood is a dangerous disease of bee broods found worldwide, caused by the Paenibacillus larvae larvae L. bacterium. In an experiment, the possibility of detecting colonies of this bacterium on MYPGP substrates (which contains yeast extract, Mueller-Hinton broth, glucose, K2HPO4, sodium pyruvate, and agar) was tested using a prototype of a multi-sensor recorder of the MCA-8 sensor signal with a matrix of six semiconductors: TGS 823, TGS 826, TGS 832, TGS 2600, TGS 2602, and TGS 2603 from Figaro. Two twin prototypes of the MCA-8 measurement device, M1 and M2, were used in the study. Each prototype was attached to two laboratory test chambers: a wooden one and a polystyrene one. For the experiment, the strain used was P. l. larvae ATCC 9545, ERIC I. On MYPGP medium, often used for laboratory diagnosis of American foulbrood, this bacterium produces small, transparent, smooth, and shiny colonies. Gas samples from over culture media of one- and two-day-old foulbrood P. l. larvae (with no colonies visible to the naked eye) and from over culture media older than 2 days (with visible bacterial colonies) were examined. In addition, the air from empty chambers was tested. The measurement time was 20 min, including a 10-min testing exposure phase and a 10-min sensor regeneration phase. The results were analyzed in two variants: without baseline correction and with baseline correction. We tested 14 classifiers and found that a prototype of a multi-sensor recorder of the MCA-8 sensor signal was capable of detecting colonies of P. l. larvae on MYPGP substrate with a 97% efficiency and could distinguish between MYPGP substrates with 1–2 days of culture, and substrates with older cultures. The efficacy of copies of the prototypes M1 and M2 was shown to differ slightly. The weighted method with Canberra metrics (Canberra.811) and kNN with Canberra and Manhattan metrics (Canberra. 1nn and manhattan.1nn) proved to be the most effective classifiers.

Comparison of In Vitro Susceptibility of Delafloxacin with Ciprofloxacin, Moxifloxacin, and other Comparator Antimicrobials Against Isolates of Nontuberculous Mycobacteria

Nontuberculous mycobacteria (NTM) infections are increasing globally. Mycobacterium avium complex (MAC) and M. abscessus complex are the most commonly reported NTM. Oral treatment options are limited, especially for the M. abscessus. We tested delafloxacin, a new oral fluoroquinolone, against 131 isolates of NTM. Delafloxacin microdilution MICs were performed as recommended by the Clinical and Laboratory Standards Institute using cation adjusted Mueller Hinton broth. The rapidly growing mycobacteria tested included: M. abscessus subsp. abscessus (16) and subsp. massiliense (5), M. chelonae (11), M. immunogenum (5), M. fortuitum group (13), M. porcinum (7), M. senegalense (7), M. mucogenicum group (5), and M. goodii (1). For the slowly growing NTM (SGM), M. avium (16), M. intracellulare (13), M. chimaera (9), M. arupense (5), M. simiae (5), M. lentiflavum (4), M. kansasii (6), and M. marinum (3) were tested. Delafloxacin was most active in vitro against M. fortuitum and M. mucogenicum groups and M. kansasii with MIC50 values of 0.12-0.5 μg/mL (MIC range 0.001-4 μg/mL) compared to ≤0.06->4 μg/mL for ciprofloxacin and ≤0.06->8 μg/mL for moxifloxacin. For other SGM (including MAC), and the M. abscessus/chelonae, the delafloxacin MIC range was 8->16 μg/mL compared to ciprofloxacin and moxifloxacin of 0.5->4 μg/mL and ≤0.06-8 μg/mL, respectively. To our knowledge, this is the first MIC study with delafloxacin to use Clinical and Laboratory Standards Institute (CLSI) recommended methods. This study illustrates the potential utility of delafloxacin in treatment of infections due to some NTM.

Novel Ferrocenyl Chalcone Derivatives as Antibacterial Agents: Is There a Solution to the Problem?

Increased infection spread is partly facilitated by reduced new drug development. Because of their antimicrobial properties, ferrocenyl chalcone derivatives were assessed in a previous study. However, dilutions of stock ferrocenyl chalcone solution with Mueller-Hinton broth (MHB) resulted in particle formation, and a colour change from deep red to dark-brown. Results of the current study confirmed particle formation, which suggested the chelation of casein hydrolysate, a component of MHB, by iron ion. After solubilisation in dimethyl sulfoxide (DMSO), each of the iodine-containing compounds, also changed from deep red to dark-brown. Mean rates of colour change (RA) in polypropylene tubes at 37oC were the highest (0.0102 ± 0.0005 ΔA/min – 0.0041 ± 0.0009 ΔA/min) while the same observed reaction in borosilicate glass tubes 21oC ± 1 were the lowest (0.0024 ± 0.0007 ΔA/min – 0.0021 ± 0.0003 ΔA/min). Antimicrobial activity of two randomly selected ferrocenyl chalcone compounds (hexyl and heptyl) was unaffected after colour change occurred (0.016 mg/ml – 0.125 mg/ml). Although these findings potentially indicate that short-term storage of antimicrobials is unaffected, further work is required to assess whether antimicrobial activity is affected by longer storage conditions.

Bee Bread Exhibits Higher Antimicrobial Potential Compared to Bee Pollen

This study aimed at investigation of the antimicrobial potential of ethanolic extracts of bee bread (BB) and bee pollen (BP) and suspensions of these products in MHB (Mueller Hinton Broth). We covered 30 samples of BP and 19 samples of BB harvested in Polish apiaries. Slightly lower activity was observed against Gram-negative bacteria compared to Gram-positive staphylococci. BB extracts exhibited higher inhibitory potential with minimum inhibitory concentration (MIC) values in the range from 2.5 to 10% (v/v) against Staphylococcus aureus ATCC 25923 and ATCC 29213. Most active BB extracts, namely, BB6, BB11 and BB19, effectively inhibited growth of clinical isolates of S. aureus (n = 9), including MRSA (methicillin resistant Staphylococcus aureus) strains (n = 3) at concentrations ranging from 2.5 to 5.0% (v/v). Minimal bactericidal concentration (MBC) values were in the same range of concentrations; however, a shift from 2.5 to 5.0% (v/v) was observed for some products. The most active BP extracts inhibited the growth of reference strains of S. aureus at a concentration of 5% (v/v). Up to the concentration of 20% (v/v) three and seven BP extracts were not able to inhibit the growth of S. aureus ATCC 29213 and S. aureus ATCC 25923 respectively. The growth of staphylococci was also importantly inhibited in suspensions of the products in MHB. No correlation between phenolic content and antimicrobial activity was observed.

Anti-Biofilm Effect of Octenidine and Polyhexanide on Uropathogenic Biofilm-Producing Bacteria

<b><i>Background:</i></b> A catheter allowing a release of antibacterial substances such as antiseptics into the bladder could be a new way of preventing biofilm formation and subsequent catheter-associated urinary tract infections. <b><i>Methods:</i></b> Minimal inhibitory and bactericidal concentration (MIC/MBC) determinations in cation-adjusted Mueller-Hinton broth and artificial urine were performed for 4 antiseptics against 3 uropathogenic biofilm producers, <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, and <i>Proteus mirabilis</i>. Furthermore, effects of octenidine and polyhexanide against catheter biofilm formation were determined by quantification of biofilm-producing bacteria. <b><i>Results:</i></b> Sodium hypochlorite showed MIC/MBC values between 200 and 800 mg/L for all strains tested. Triclosan was efficient against <i>E. coli</i> and <i>P. mirabilis</i> (MIC ≤2.98 mg/L) but ineffective against <i>P. aeruginosa</i>. Octenidine and polyhexanide showed antibacterial activity against all 3 species tested (MIC 1.95–7.8 and 3.9–31.25 mg/L). Both octenidine and polyhexanide were able to prevent biofilm formation on catheter segments in a concentration dependent manner. Furthermore, adding 250 mg/L of each biocide disrupted biofilms formed by <i>E. coli</i> and <i>P. mirabilis</i>, whereas even 500 mg/L was not sufficient to completely destroy <i>P. aeruginosa</i> biofilms. <b><i>Conclusion:</i></b> Octenidine- and polyhexanide-containing antiseptics showed a broad effect against typical uropathogenic biofilm producers even in high dilutions. This study provides a basis for further investigation of the potential of octenidine and polyhexanide as prophylaxis or treatment of catheter biofilms.