The potency of Tigecycline in the Sensititre plates

Michel Rapallinim.l.b.a.rapallini@nvwa.nl

Content• The Netherlands Food and

Consumer Product Safety Authority (NVWA)

• Antimicrobial susceptibilitytesting at the NVWA

• Tigecycline resistantEnterococci 2014

• Stability of Tigecycline

• Conclusion

The Netherlands Food and Consumer Product Safety Authority (NVWA)

The task and activities of the NVWA:To protect human and animal health by monitoring food and consumer products.

The Authority controls the whole production chain, from raw materials and processing aids to end products and consumption.3

Antimicrobial susceptibility testing 2014Strains: Panels Sensititre: Samples 2014 (n):Salmonella EUVSEC 291E. coli EUVSEC 1382 Enterococci EUVENC 1015Campylobacter EUCAMP2 402ESBL EUVSEC2 686

Equipment:

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Antimicrobial susceptibility testing NVWAThe results are annually reported to the EFSA and published in the MARAN(Monitoring of Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands)

MARAN published by the CVI (Central Veterinary Institute) based on:• Total sales data and animal specific usage of antimicrobial agents in animal

husbandry.• Development of antimicrobial resistance in bacteria of animal origin.• Relevance to public health.

In collaboration with:

Netherlands Veterinary Medicines Authority NVWA RIVM

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Tigecycline resistant Enterococci 2014Introduction panel EUVENC January 2014

ANTIMICROBICSVAN VancomycinTEI TeicoplaninSYN Quinupristin /

dalfopristinTET TetracyclineDAP DaptomycinCIP CiprofloxacinERY ErythromycinTGC TigecyclineLZD LinezolidGEN GentamicinAMP AmpicillinCHL ChloramphenicolPOS Positive Control

*Resistant=>0.25 Tigecycline

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VAN128

TEI64

SYN64

TET128

DAP32

CIP16

ERY128

TGC4

LZD64

GEN1024

AMP64

CHL128

VAN64

TEI32

SYN32

TET64

DAP16

CIP8

ERY64

TGC2

LZD32

GEN512

AMP32

CHL64

VAN32

TEI16

SYN16

TET32

DAP8

CIP4

ERY32

TGC1

LZD16

GEN256

AMP16

CHL32

VAN16

TEI8

SYN8

TET16

DAP4

CIP2

ERY16

TGC0.5

LZD8

GEN128

AMP8

CHL16

VAN8

TEI4

SYN4

TET8

DAP2

CIP1

ERY8

TGC*0.25

LZD4

GEN64

AMP4

CHL8

VAN4

TEI2

SYN2

TET4

DAP1

CIP0.5

ERY4

TGC0.12

LZD2

GEN32

AMP2

CHL4

VAN2

TEI1

SYN1

TET2

DAP0.5

CIP0.25

ERY2

TGC0.06

LZD1

GEN16

AMP1

POSCON

VAN1

TEI0.5

SYN0.5

TET1

DAP0.25

CIP0.12

ERY1

TGC0.03

LZD0.5

GEN8

AMP0.5

POSCON

Tigecycline resistant Enterococci 2014 (n)

Enterococci examined: 1015Resistant for tigecycline (MIC>0.25): 263 (26%)

E. feacalis ATCC 29212: 31Resistant for tigecycline (MIC>0.25): 7 (23%)

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Batch EUVENCpanels used

Enterococci examined(n)

TIG Resistant (MIC>0.25)(n)

B3513 445 33B4255 458 165B4423 112 65

Total 1015 263

Stability of TigecyclineAntimicrob Agents Chemother. 2005 Sep; 49(9): 3903–3909. PMCID:PMC1195415Tigecycline MIC Testing by Broth Dilution Requires Use of Fresh Medium or Addition of the BiocatalyticOxygen-Reducing Reagent Oxyrase To Standardize the Test Method“It was previously established that tigecycline is susceptible to oxidative degradation in a solution”

PLoS One. 2014; 9(5): e95281.Published online 2014 May 28. doi:10.1371 PMCID:PMC4037176A Novel Formulation of Tigecycline Has Enhanced Stability and Sustained Antibacterial and AntileukemicActivity“In this study, we identified that the degradation of tigecycline after reconstitution is accelerated by exposure to light. It is well known that tigecycline degradation is oxygen-dependant”

J. of Chromatographic Sci. July 24, 2012. 192-9.doi: 10.1093 PMID:22832547Validation of a Stability-Indicating RP-LC Method for the Determination of Tigecycline in Lyophilized Powder“confirming the high reactivity of the tigecycline with oxygen, made possible by the low stability of this drug” “Under the photolytic condition, a significant decrease of the area was also observed”

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Stability of TigecyclineWe examined whether practical preparations could be the reason for these unexpected results.

Frequently, all EUVENC plates were stacked with the protective cover already removed for some time (in some cases 4 to 5 hours prior to analysis).

We suspected that this could explain theunexpected Tigecycline resistant isolates.

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Stability of Tigecycline

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SAMPLE NO. ORGANISM EUVENC panel results TGS NVWA 2014 EUVENC panel results TGC CVIpanel cover

removed 4 hours prior to analysispanel cover

removed just prior to analysisMIC (µg/ml) S / R MIC (µg/ml) S / R MIC (µg/ml) S / R

EF0464 E.FAEM = 0.06 S = 0.25 S = 0.06 SEF0612 E.FAEM = 0.06 S = 0.12 S = 0.06 SEF0846 E.FAEM = 0.06 S = 0.12 S = 0.06 SEF0906 E.FAEM = 0.06 S = 0.06 S = 0.06 SEF0914 E.FAEM = 0.06 S = 0.06 S = 0.06 SEF0482 E.FAEM = 0.12 S = 0.25 S = 0.06 SEF0605 E.FAEL = 0.12 S = 0.25 S = 0.12 SEF0611 E.FAEM = 0.12 S = 0.12 S = 0.12 SEF0616 E.FAEM = 0.12 S = 0.12 S = 0.12 SEF0646 E.FAEM = 0.12 S = 0.06 S = 0.06 SEF0753 E.FAEM = 0.12 S = 0.12 S = 0.06 SEF0863 E.FAEM = 0.12 S = 0.12 S = 0.06 SEF0898 E.FAEL = 0.12 S = 0.25 S = 0.12 SEF0902 E.FAEM = 0.12 S = 0.12 S = 0.12 SEF0442 E.FAEM = 0.25 S = 0.25 S = 0.25 SEF0527 E.FAEL = 0.25 S = 0.25 S = 0.12 SEF0619 E.FAEM = 0.25 S = 0.12 S = 0.06 SEF0633 E.FAEM = 0.25 S = 0.12 S = 0.06 SEF0835 E.FAEM = 0.25 S = 0.12 S = 0.06 SEF1443 E.FAEM = 0.25 S = 0.06 S = 0.06 SEF1446 E.FAEM = 0.25 S = 0.12 S = 0.06 SEF0592 E.FAEL = 0.5 R = 0.12 S = 0.12 SEF0821 E.FAEL = 0.5 R = 0.12 S = 0.06 SEF1501 E.FAEM = 0.5 R = 0.12 S = 0.12 SEF1502 E.FAEM = 0.5 R = 0.12 S = 0.06 S

Stability of Tigecycline

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SAMPLE NO. ORGANISM EUVENC panel results TGS NVWA 2014 EUVENC panel results TGC CVIpanel cover

removed 4 hours prior to analysispanel cover

removed just prior to analysisMIC (µg/ml) S / R MIC (µg/ml) S / R MIC (µg/ml) S / R

EF0464 E.FAEM = 1 R = 0.06 S = 0.12 SEF0612 E.FAEM = 1 R = 0.06 S = 0.06 SEF0846 E.FAEM = 1 R = 0.25 S = 0.12 SEF0906 E.FAEM = 1 R = 0.06 S = 0.06 SEF0914 E.FAEM = 1 R = 0.25 S = 0.06 SEF0482 E.FAEM = 1 R = 0.12 S = 0.06 SEF0605 E.FAEL = 1 R = 0.12 S = 0.25 SEF0611 E.FAEM = 1 R = 0.12 S = 0.06 SEF0616 E.FAEM = 1 R = 0.12 S = 0.12 SEF0646 E.FAEM = 1 R = 0.12 S = 0.12 SEF0753 E.FAEM = 1 R = 0.25 S = 0.12 SEF0863 E.FAEM = 1 R = 0.06 S = 0.06 SEF0898 E.FAEL = 1 R = 0.12 S = 0.25 SEF0902 E.FAEM = 1 R = 0.12 S = 0.25 SEF0442 E.FAEM = 1 R = 0.12 S = 0.12 SEF0527 E.FAEL = 1 R = 0.12 S = 0.12 SEF0619 E.FAEM = 1 R = 0.12 S = 0.25 SEF0633 E.FAEM = 1 R = 0.12 S = 0.25 SEF0835 E.FAEM = 1 R = 0.25 S = 0.12 SEF1443 E.FAEM = 1 R = 0.25 S = 0.12 SEF1446 E.FAEM = 1 R = 0.12 S = 0.12 SEF0592 E.FAEL = 1 R = 0.12 S = 0.12 SEF0821 E.FAEL = 1 R = 0.12 S = 0.12 SEF1501 E.FAEM = 1 R = 0.12 S = 0.12 SEF1502 E.FAEM = 1 R = 0.12 S = 0.06 S

Stability of Tigecycline

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ConclusionThese results suggest (in our case) that:

• The prolonged exposure of Tigecycline to light and air caused photo-oxidation and thus Tigecycline degradation.

• After removing the panel cover prior to analysis, and keeping the panel prior to incubation in the dark, no more Tigecycline resistant Enterococci were identified.

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Advice

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Stay on the darkside with your panels

ACKNOWLEDGEMENT NVWA:Heymans, dr. R. (Raymond)Wit, ing. B. (Ben)Nap, I.D. (Irma)Nabuurs, ing. F.C.P. (Fiona)Sijben, F.H.G. (Frank)Nendos, N. (Nico)Engelen, R.J.K. van (Ruud)Smit, C. (Corry)

CVI:Veldman, dr. T. (Kees)15