In vitro activity of dalbavancin against Neisseria gonorrhoeae and development of a broth microdilution method

Correspondence to:

Laura M. Koeth

Laboratory Specialists, Inc.

1651 A Crossings Parkway

Westlake, OH 44145

lkoeth@labspec.org

Ph: 440-835-4458 Laura M. Koeth, Jeanna Fisher

Laboratory Specialists, Inc, Westlake, OH

1. Clinical and Laboratory Standards Institute. 2012. Methods for Dilution Antimicrobial Susceptibility Tests for

Bacteria that Grow Aerobically. 9th ed. Approved standard, CLSI M7-09, Wayne, PA.

2. Koeth, LM, Windau AR, Goldstein BP. 2012. Comparison of Broth Microdilution and Agar Dilution Dalbavancin

and Vancomycin MIC results for 42 Aerobic Gram-positive Bacteria. ASM General Meeting . Poster # 123.

3. Clinical and Laboratory Standards Institute. 2013. Performance Standards for Antimicrobial Susceptibility

Testing. Approved Standard – 23th Edition. CLSI document M100-23 Wayne, PA:

Currently agar dilution is the recommended MIC method for Neisseria gonorrhoeae (1).

However, agar dilution (AD) methods are not typically used by most clinical laboratories. In

the case of dalbavancin, the AD method is not recommended because MIC results are 2-4

dilutions higher than MIC results using a broth + 0.002% polysorbate 80 (P-80) method (2).

Prior testing with dalbavancin and N. gonorrhoeae was performed with GC broth (GCB) +

0.002%. However, a precipitate was formed with the addition of the surfactant to the media

and endpoint determination was not possible due to the turbidity of the media. This study

was performed to identify other broths and testing conditions for testing of dalbavancin

against N. gonorrhoeae. The results from 2 AD and broth microdilution (BMD) MIC

comparative studies using a modified GCB for a set of challenge strains and growth curves

for the QC strain in modified GCB and another enriched broth (MTGE) is presented.

Growth Curves of N. gonorrhoeae ATCC 49226 in 3 Media:

MIC Study 1 and 2 Organisms:

Figure 1: Dalbavancin MIC Results for 24 N.gonorrhoeae :

AD and 48 Hour BMD in 5% CO2

• The addition of 0.002% P-80 to modified GC broth provides N.

gonorrhoeae dalbavancin MIC results that are on average 3 dilutions

lower compared to AD results

•The incidence of no growth with the broth testing in this study

indicates a need for further development to determine the optimal broth

and incubation conditions.

Objective: As antimicrobial resistance rates increase among Neisseria gonorrhoeae, it is

important to investigate other treatment alternatives and also to provide a methodology of MIC

testing that will be more adaptable to a routine clinical laboratory. This study was performed

to determine the in vitro activity of dalbavancin against a challenge set of Neisseria

gonorrhoeae and to initiate development of a broth microdilution method for testing of Neisseria

gonorrhoeae. Method: Dalbavancin, ceftriaxone and ciprofloxacin BMD and AD MIC results

were obtained for a set of 31 N. gonorrhoeae (11 susceptible strains including QC strain ATCC

49226 and 20 resistant strains) and QC S. aureus ATCC 29212. The CLSI recommended MIC

procedure for testing Neisseria gonorrhoeae is an agar dilution (AD) methodology. Based on

prior studies, which have shown significantly higher dalbavancin MICs with AD, a broth

microdilution method (BMD) with addition of 0.002% polysorbate 80 (P-80) is suggested for

testing Neisseria gonorrheae against dalbavancin. A modified GC broth (substitution of

glucose instead of starch) + 0.002% P80 was used for testing of dalbavancin and GC broth was

used for the two comparator agents. All MIC plates were incubated in CO2 and results were

read after 48 hours.

Results: Dalbavancin BMD MIC results were 0.12-1 µg/mL compared to AD MIC results of 1-4

µg/mL. There were 7 strains that did not grow in the modified GC broth. All strains were

susceptible to ceftriaxone (<0.06 µg/mL). A total of 9 strains were resistant to ciprofloxacin

(>0.5 µg/mL) and remaining 12 strains were susceptible (≤0.06 µg/mL). The dalbavancin BMD

MIC for N. gonorrhoeae ATCC 49226 was 0.5-1 µg/mL and for S. aureus ATCC 29213 was

0.06-0.12 µg/mL. The dalbavancin MIC distribution (number at each MIC) for 24 strains by MIC

method is shown in the table.

Conclusion: The in vitro activity of dalbavancin against this challenge set of Neisseria

gonorrhoeae by BMD is an indication of possible in vivo activity that will require further

investigation. As a result of lack of growth with some strains in the modified broth, additional

development is necessary using the broth methodology for dalbavancin.

Method MIC (µg/mL)

0.12 0.25 0.5 1 2 4

BMD 2 13 7 2

AD 4 14 6

Table 1: Frequency of Dilution Difference by Drug and Broth Method

Poster Number 255

GC Broth 15 g protease peptone 3

1 g soluble starch

4 g K2HPO4

1 g KH2PO4

5 g NaCl

1 L dH20

Aseptically supplement after

autoclaving and cooling with 10

mLs of IsoVitalex

Modified GC Broth Same as GC Broth except 1 g

dextrose is used instead of soluble

starch and 1 mL 2% P-80 is added

aseptically with the IsoVitalex.

MTGE Broth Protein Mixture, 10.0 g

Yeast Extract, 5.0 g

Sodium Bicarbonate, 5.0 g

Sodium Format15e, 0.5 g

Sodium Fumarate, 1.0 g

Sodium Succinate, 0.5 g

Potassium Phosphate, dibasic, 2.0 g

Sodium Chloride, 5.0 g

Magnesium Sulfate, 0.1 g

Dextrose, 1.0 g

Volatile Fatty Acid Mix, 3.0 ml

Vitamin K1 (1% soln), 1.0 ml

Sodium Pyruvate, 0.8 g

Thiamine Pyrophosphate, 0.025 g

L-Cysteine, 0.5 g

Serum, 50.0 ml

Distilled Water, 940.0 ml

(Anaerobe Systems, Morgan Hill, CA)

IsoVitalex

Approximate Formula per L

Purified Water

Vitamin B12 0.01 g

L-Glutamine 10.0 g

Adenine 1.0 g

Guanine

Hydrochloride 0.03 g

p-Aminobenzoic Acid 0.013 g

Nicotinamide Adenine

Dinucleotide 0.25 g

Thiamine

Pyrophosphate 0.1 g

Ferric Nitrate 0.02 g

Thiamine

Hydrochloride 0.003 g

L-Cysteine

Hydrochloride 25.9 g

L-Cystine 1.1 g

Dextrose 100.0 g

(BBL, Becton Dickinson, Sparks,

MD)

Broth Ingredients:

• Strain #1-20: challenge strains provided by University of Washington; included 18 that

were resistant to ciprofloxacin, some which had high cefpodoxime or cefixime MICs and

others with additional resistance to penicillin and tetracycline.

• Strain #21-30: typical mix of clinical strains from frozen stock provided by University

Hospital of Cleveland; only one strain was resistant to ciprofloxacin.

• QC strains: N. gonorrhoeae ATCC 49226 and S. aureus ATCC 29213, CLSI expected

ranges (3)

MIC Study 1 and 2 Design:

Bacterial suspension was made similar to MIC method in three different media (GCB, mGCB

and MTGE broth) and inoculated into 96 well plates also containing the three media. Initial

colony counts were performed and plates were sealed with standard plate seal, perforated

plate seal or plate cover with plate placed in container with damp towel. Colony counts were

also performed after 24 and 48 hours incubation at 35ºC in 5% CO2.

This study was sponsored by a grant from Durata Therapeutics, Inc,.

Figure 3: Growth Curve for N. gonorrhoeae ATCC 49226 in 3 Media

(a) GC Broth (b) modified GC Broth (c) MTGE Anaerobic Enrichment

Broth

•BMD results could not be determined at 20 and 24 hours (no visible growth).

•Dalbavancin BMD MICs (48 hours in 5% CO2) were 0.125-1 µg/mL and were 1-4 dilutions lower

than AD MICs, with majority 3 dilutions lower (Fig. 1 and Table 1)

•The incidence of no growth (in growth control wells) for plates incubated 48 hours in 5% CO2

varied from 6 to 11 strains (Table 1). No growth occurred in dalbavancin BMD plates with the

same 6 strains in both studies (all had AD MIC results of 1 µg/mL and 5/6 were ciprofloxacin

susceptible strains).

•Ceftriaxone BMD MICs were 0.002-0.03 µg/mL (48 hours in 5% CO2) and were 1-2 dilutions

lower to 1 dilution higher than AD MIC results, with majority at 1-2 dilutions lower (Fig. 2)

•Ciprofloxacin BMD MICs were 0.002-0.06 µg/mL for 11 strains and >0.5 µg/mL for 19 strains.

•Dalbavancin S. aureus ATCC 25923 results were 4 µg/mL by AD (GC media) and 0.03-0.12

µg/mL (mGCB). Ciprofloxacin and ceftriaxone QC results were within expected CLSI AD ranges

for N. gonorrhoeae ATCC 49226 and BMD ranges for S. aureus ATCC 25923.

AD and BMD MIC Studies:

N. gonorrhoeae ATCC 49226 Growth Curves (Fig. 3):

• GC Broth: Growth increased from 0 hours to 24 hours by 2-2.5 log(10) for each

broth/condition, with exception of plates in container (increase was slightly < 2 log)

• Modified GC Broth : Growth remained stationery from 24 to 48 hours with perforated

sealed plates and decreased slightly with non-perforated sealed plates and plates in container

•MTGE Broth: Growth continued to increase from 24 to 48 hours with both perforated and

non-perforated sealed plates

Study Number 1 2

MIC Method Agar Dilution (1) Broth Microdilution Broth Microdilution

Media GC Agar GC Broth mGCB mGCB, w/3 dextrose levels*

Incubation Conditions 5% CO2 Ambient & 5% CO2, sealed plates 5% CO2, sealed plates

Incubation Time 24 hrs 20, 24, 48 hrs 48 hrs

Dalbavancin √ √ √

Ceftriaxone √ √

Ciprofloxacin √

mGCB - modified GC broth (GC broth with 0.002% P80 and starch replaced with dextrose)

*0.5 g/L, 1 g/L and 2 g/L of dextrose

-7 -6 -5 -4 -3 -2 -1 0 1 2 3 NG

Dalbavancin (mGCB BMD - AD)

Study 1 Ambient 2 6 11 2 9

Study 1 5% CO2 4 12 7 7

Study 2 5% CO2 6 7 9 2 6

Study 2 2X Dextrose 1 1 4 10 5 3 6

Study 2 ½X Dextrose 8 11 3 2 6

Ceftriaxone (GC BMD - AD)

Study 1 Ambient 2 10 2 2 1 13

Study 1 5% CO2 7 7 3 2 11

NG - No Growth