effects of Microplate Type in Broth additives on Microdilution.pptx

effects of Microplate Type in Broth additives on Microdilution.pptx

1. Objective They examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in media alone and in media supplemented with commonly used additives Tween-80, lysed horse blood, and 50% human serum.

2. Materials and methods Gram-positive bacteria Staphylococcus aureus MSSA ATCC 25923 MRSA ATCC 43300 VISA ATCC 700698 Streptococcus pneumoniae ATCC 33400 MDR ATCC 700677 Enterococcus faecalis (ATCC 29212) Streptococcus pyogenes (ATCC 12344), Gram- negative bacteria Escherichia coli (ATCC 25922) Klebsiella pneumoniae (ATCC 13883) Pseudomonas aeruginosa (ATCC 10145) Acinetobacter baumannii (ATCC 19606)

3. Materials and methods Gram-positive bacteria Vancomycin Telavancyn Dalbavancin Ciprofloxacin Gram- negative bacteria Colistin Timehoprim Polymyxin B Penicillin G Rifampin Antibiotics were selected to include those previously reported to exhibit plate- or surfactant-based MIC variations , as well as examples expected to not show an effect . Antibiotics MCC223, MCC310 and MCC520 Drug discovery program (vancomycin derivates) Vancapticins Additives Polysorbate 80 ( Tween 80) Human serum Lysed horse blood

4. Materials and methods The plate types 96-well Corning flat-bottom PS untreated Costar 3370 Corning flat-bottom TC surface COR 3628 Corning flat-bottom NBS surface COR 3641 Thermo Electric flat-bottom untreated PS Nunc-442404 Thermo Electric flat-bottom TC surface Nunc-167008 Thermo Electric U-bottom TC surface Nunc-163320 Trek Diagnostics untreated H511A PS : polistyrene , TC: Tissue culture- treated , NSB: non- binding surface

5. MIC determination via broth microdilution assay. Duplicate Positive and negative controls in each plate Standars ranged from 64 g/ml to 0.03 g/ml and compounds from 8 g/ml to 0.003 g/ml G+ and G- bacteria were cultured in MHB with and without 0.002% Tween at 37°C overnight. For experiments in the presence of surfactant 2% beractant (25 mg/ml) For experiments in the presence of serum, mixture of 50% of human serum along with 50% MHB

6. Experimental Design Frist experiments Seven different PS- based plate Types 1 G+ vs. seven antibiotics 1 G- vs. seven antibiotics MHB and in MHB + 0.002% T80 to see if the plate type could obviate surfactant .

7. Second experiments : Whether differences between plates seen in the first experiments were consistent across bacterial strains. Three plate types (Corning) Same sets of antibiotics Six additional G+ and three G- MHB medium with and without T80. Experimental Design

8. Experimental Design Third experiments Compared the effects of different additive used to assess the effectiveness of antibiotics under physiological conditions 50% human serum 2% lung surfactant. These were done in four plate types Two PS untreated TC treated NBS treated Three broth MHB, MHB with added 0.002% T80 MHB with added 2% LHB

9. Results Experiment 1, initial plate comparison with or without Tween 80. Seven different plate types Gram-positive (methicillin-resistant Staphylococcus aureus [MRSA] ATCC 43300) Gram-negative ( Escherichia coli ATCC 25922) MHB with and without 0.002% T80

10. Low protein binding Nonliphofilic antibiotics Little variations More portent activity The nontreated plates showed varied effects depending on the manufacturer, while the TC-coated plates uniformly showed decreased antibiotic activity.

11. Little variations PS plates more potent values

12. Results Experiment 2, plate plus Tween 80 comparison versus expanded set of bacteria. Plate variations (PS, TC, and NBS) vs. different bacteria seven antibiotics against either seven Gram-positive or four Gram-negative bacteria.

13. Variations depends on the type of antibiotic and the plate used usually less potent in TC plates without T80 than with T80

14. Results Experiment 3, plate plus additives comparison In assessing the activity of potential antibiotics, it is important to conduct assays in the presence of biological components that the antibiotics will encounter in the human body. Four plate types (PS, TC, and NBS from Corning, and PS from Trek) MHB, MHB plus 50% human serum (HS), and MHB plus 2% artificial lung surfactant (LS). with no additive, and with T80, 2% lysed horse blood (LHB)

15. Little variations Dalvavancyn was inactivated in presence of 50% HS in all type plates NBS plates gave the most potent activity for all lipoglycopeptides but with reductions in activity when T80 or LHB was added. Variations depends on the type of antibiotic , the plate used médium and additive .

16. Ciprofloxacin , oxacillin , penicillin G, trimethoprim, and rifampin showed little variation even with HS or LS additives .

17. Conclution There are subtle variations depending on the antibiotic, plate type, and additives employed. They observed very significant differences in measured MICs for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, Microtiter plate types and any additives should be specified when reporting broth dilution MIC values, as results can vary dramatically for some classes of antibiotics.

18. Objetive T o evaluate different methods to try to improve the discrimination of Shh and Shn isolated from blood cultures, including their susceptibility to oxacillin and vancomycin and clonal profiles.

19. Metohodology Forty-nine S. hominis isolates, obtained from blood cultures at a tertiary care hospital located in Rio de Janeiro, Brazil , n=17, 1998-2002 and n= 32, 2005-2009 Microscan ( frist identification ) SDS-PAGE MALDI-TOF Partial sequencing of the 16S rDNA gene, Subspecies identification Acid production from D- trehalose (SHH) Novobiocin disk difusión broth microdilution (0.250-16 μ g/ml) Minimum inhibitory concentrations (MICs) to oxacillin and vancomycin broth microdilution Genomic diversity PFGE typing mecA gene detection and SCC mec typing

20. Results Forty-nine S. hominis isolates were initially identified at subspecies level by phenotypic tests by using the MicroScan WalkAway ® automation and novobiocin susceptibility tests. Then SDS-PAGE, MALDI-TOF and sequencing were applied All methods were concordant to identify an amount of 28 (57%) isolates, 8 S. hominis hominis ( Shh ) and 20 S. hominis novobiosepticus ( Shn ) Group I and II

21. Results The other 21 (43%) isolates could not be properly discriminated, and by comparing all three methodologies. They were indentified as SHH by MicroScan , however , they were identified as SHN by SDS-PAGE and MALDI-TOF 19 of these 21 isolates (Group III) were sensitive to the novobiocin disk a Low MIC values, ranging from <0.25 to 1.0 μg /ml The two remaining isolates (Group IV) were resistant to novobiocin (MICs of 8.0 and 16 μ g/ mL

22. Results SDS-PAGE common regions ( similarity index 90.4%) SHN: specific proteins 49 kDa y 85kDa. SHH: specific proteins 15 kDa , 26 kDa , 37 kDa . MALDI-TOF T he mass spectrometry analysis also showed a distinct protein profile spectrum Peaks betwwen 25-52 kDa . Sequencing Sigle Nucleotide Polimorfism (SNP 112: G A) of rDNA 16S amplicon , Thus, based on these data, all the 21 misidentified isolates were identified as Shn

23. results Gene mec A Out of the 41 isolates identified as Shn 92.7% mec A (+) Out of the 8 isolates of SHH 12.5% mec A (+) Vancomycin and Oxacillin susceptibility O xacillin MIC (classification by MALDI-TOF and SDS-PAGE ) SHH: <0.125 to 4 μg /mL SHN: <0.125 to >256 μg /mL Vancomycin MIC (classification by MALDI-TOF and SDS-PAGE ) SHN: 0.5 to 2 μg /mL SHN: ≤ 1 μg / mL. PFGE no clonal relationship between S. hominis subspecies isolates

24. Conclution In conclusion, their results showed that the phenotypical methods normally used for discrimination of S. hominis subspecies are not reliable. Hence, they suggest that protein profile analysis and/or MALDI-TOF MS analysis might be complementary and useful tools to discriminate these subspecies more accurately, contributing to understanding its clinical epidemiology .

25. Novel potential biomarker mass peaks for rapid identification of Staphylococcus hominis  subsp. novobiosepticus by MALDI-TOF MS Objetivo: buscar potenciales marcadores proteicos para la rápida diferenciación de las subespecies de S. hominis .

26. Materiales y Métodos 18 SHN y 58 SHH recuperados de sangre y LCR 2006-2018 Identificación por MALDI-TOF Subespecies Disco de novobiocina (5 μ g/ml) Microdilución (1.6 μ g/ml) Comparación de los perfiles proteicos ClinProTools SHH vs SHN

27. Resultados 28 picos estadísticamente significativos entre las subespecies. Table 1. Statistically significant potential peaks of SHH and SHN subspecies differentiation SHN n=18 SHH n=58 Peak AUC Present Absent Present Absent 2166.12 0.94 2 16 45 13 2273.61 0.95 1 17 47 11 2289.51 0.95 1 17 53 5 2582.72 0.93 15 3 1 57 2619.07 0.95 18 43 15 2823.13 0.92 15 3 7 51 3206.02 0.95 18 45 13 3323.25 0.98 18 6 52 4069.79 0.97 18 14 44 4339.64 0.97 18 45 13 4355.91 0.97 18 43 15 4831.67 0.97 8 10 11 47 5169.4 0.97 15 3 2 56 5242.27 0.92 18 48 10 5953.28 0.96 18 42 16 6648.56 0.98 17 1 5 53 6873.7 0.97 18 47 11 7560.41 0.95 14 4 2 56 7755.47 0.95 16 2 6 52 7921.54 0.94 13 5 58 7959.79 0.98 15 3 2 56 7981.54 0.94 14 4 1 57 8220.82 0.98 16 2 2 56 8493.62 0.98 18 3 55 8607.27 0.92 15 3 11 47 9660.02 0.96 18 9 49 10199.49 0.97 17 1 4 54 11120.68 0.94 16 2 6 52 15114.27 0.98 14 4 1 57 PAD: p- value Anderson-Darling, PTTA: p- value t-test/ANOVA, PWKW: p- value Wilcoxon/ Krustal -Wallis, AUC: area under curve, SHH: S. hominis subsp hominis, SHN: S. hominis subsp novobiosepticus Resultados parciales

28. Resultados

29. Conclusión Se encontraron 29 picos con potencial de biomarcadores para la rápida diferenciación de S. hominis subsp . novobiosepticus por MALDI-TOF MS. Los valores de rendimiento de los biomarcadores oscilaron entre 0,92 y 0,98 (AUC).

30. Estructura planeada Objetivo: Diferencias entre las subespecies de S. hominis Comparar las metodologías Micro dilución, disco y análisis ClinProTools Campos y secuenciación n: 18 SHN MIC y disco 230 aislamientos de S. hominis Análisis ClinProTools SHH y SHN Incluir las cepas control SHH ATCC 27844 SHN ATCC 700236 (no se tiene)

31. PROPUESTA Búsqueda de potenciales biomarcadores para predecir la resistencia a LZD en SHN y SHH usando ClinProTools . Se tienen 11 aislamientos resistentes a Lzd 10 Resistentes SHN (10/18) 1 resistente SHH (1/58) Detectar genes involucrados en la resistencia ( cfr ) SDS-PAGE patrón proteico Relación pico-proteína Purificación e identificación de las proteínas HPLC MS / LC MS Incluir otras especies de ECN (tesis maestría Adrián) S. epidermidis 3 resistentes Lzd S. haemolyticus 2 resistentes Lzd Incluir aislamientos resistentes a lzd de Guadalajara. (n=24) Confirmar resistencia a lzd

32. Análisis ClinProTools de SHN LZD Sensibles n= 8 Resistentes n= 10 25 picos estadísticamente significativos