effects of Microplate Type in Broth additives on Microdilution.pptx

2. 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.

3. 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)

4. 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

5. 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

6.  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

7. 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.

8. 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

9. 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

10. 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

11. 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.

13. Little variations
PS plates more potent
values

15. 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.

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

17. 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)

18. 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.

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

22. 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.

24. OBJETIVE
 To 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.

25. 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 μg/ml)
 Minimum inhibitory concentrations (MICs) to oxacillin and vancomycin
 broth microdilution
 Genomic diversity PFGE typing
 mecA gene detection and SCCmec typing

26. 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

28. 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 μg/mL

30. 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
The 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

32. RESULTS
 Gene mecA
 Out of the 41 isolates identified as Shn
 92.7% mecA (+)
 Out of the 8 isolates of SHH
 12.5% mecA (+)
 Vancomycin and Oxacillin susceptibility
 Oxacillin 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

33. 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.