Objective:

To evaluate the performance of Brilliance ESBL® (OX; Oxoid) selective chromogenic agar

for the detection and the presumptive identification of extended-spectrum beta-

lactamases (ESBL)-producing Enterobacteriaceae (EB).

Methods:

In a preliminary study, we challenged a collection of 200 Gram-negative bacterial

including 156 EB strains with well-defined resistance mechanisms against OX and ChromID

ESBL® (BM; bioMérieux) chromogenic agar to evaluate the growth selectivity and the

chromogenic features of the media. In a second part, 528 clinical samples (including 344

fecal specimens) obtained from 424 ambulatory and hospitalized patients were plated

onto OX, BM and MacConkey agar to which a disk of ceftazidime was added (MCC) for the

screening of ESBL-producing EB. All colonies growing on any of the three media after full

24-hour incubation were identified and tested for susceptibility by Phoenix System

(Becton-Dickinson). ESBL confirmation was performed by combination disks method.

Characterization of resistance mechanisms was determined by PCR of TEM, SHV, CTX-M,

OXA, and AmpC genes with amplicon sequencing.

Results:

Of the 156 EB isolates from the collection isolates, all 98 ESBL producers were detected

but 50 strains harbouring various non-ESBL resistance mechanisms were also recovered on

both OX and BM; 8 fully susceptible isolates did not grow on any of the two selective

media. Of the 528 clinical samples screened, 144 (27%) yielded growth on at least one of

the three media. A total of 182 isolates including 109 (60%) EB were recovered and 70 of

these (from 59 specimens) were confirmed as ESBL-producing isolates. The sensitivities

were 74.6%, 94.9% and 94.9% for MCC, BM and OX respectively. The specificities

calculated for the ESBL-negative samples reached 94.9%, 95.5% and 95.7% for MCC, BM

and OX respectively when only chromogenic enterobacterial colonies were considered on

the two chromogenic media.

Conclusions:

OX and BM yielded comparable performances and had a higher sensitivity than MCC for

the detection of ESBL-producing EB from clinical specimens. The high negative predictive

value (99.3%) found for OX suggested that this medium could represent an excellent

screening tool for rapid exclusion of carriage of ESBL producers. The selection of

enterobacterial isolates only for ESBL confirmation based on chromogenic features could

limit the unnecessary workout.

Revised abstract

Evaluation of Brilliance ESBLTM, a Novel Chromogenic Agar for the Detection

of Extended-Spectrum Beta-Lactamases Producing Enterobacteriaceae

Huang T-D,1,2 Bogaerts P,2 Berhin C,2 Guisset A,2 Glupczynski Y2

Conclusions

Introduction

References

1 Laboratory of Microbiology, Cliniques Universitaires U.C.L. St-Luc, Brussels, Belgium2 Laboratory of Microbiology, Cliniques Universitaires U.C.L. Mont-Godinne, Yvoir, Belgium

Mailing address:

T-D Daniel Huang

Laboratoire de Microbiologie

Cliniques Universitaires Mont-Godinne UCL

5530 Yvoir, Belgium

E-mail: te-din.huang@uclouvain.be

Poster

P638

Methods (challenge strains)

Results (clinical samples)

Results

Results (challenge strains)

The spread of ESBLs in Gram-negative bacteria represents a

major therapeutic challenge either in hospitals or in a community

setting (1).

The use of surveillance cultures or of targeted screening for

ESBL producers in high-risk patients or units (e.g. in intensive care

units) has been advocated to prevent or to control outbreaks of

nosocomial infections caused by these organisms (2, 3).

In this study, we aimed to evaluate the performance of the

prototype chromogenic selective medium Brilliance™ ESBL (OX;

Oxoid, Basingstoke, United Kingdom) as compared to another

commercialized medium, (ChromID ESBL agar (BM); bioMérieux,

Marcy l’Etoile, France), which had been assessed in a previous

validation study (4), for their ability to detect and presumptively

identify ESBL-producing Enterobacteriaceae. The study was split

into two phases: testing a challenge collection (first part) and

direct inoculation of clinical samples (second part).

Methods (clinical samples)

The challenge set of 200 Gram negative isolates selected based on the

diversity of their resistance mechanisms from the collection of clinical

bacterial isolates of the bacteriology laboratory of the Cliniques UCL de

Mont-Godinne were tested for their ability to grow on the two

chromogenic media.

All strains were thawed from -70°C and subculture isolates were

suspended in saline than homogenized to a density of 0.5 McFarland. A

10-µl inoculum of this suspension was inoculated onto selective

chromogenic agars (OX and BM), and on MacConkey plates (MC; Oxoid)

used as growth controls. All plates were incubated at 35°C in ambient

air atmosphere for 24 h before reading.

The study was performed at the Cliniques Universitaires St-Luc in

Brussels from February 2009 to April 2009. A total of 528 samples obtained

from 424 patients (25% ambulatory and 75% hospitalized) were processed

and screened, including 344 fecal samples, 134 respiratory tract samples

and 50 samples of miscellaneous origins.

Each specimen was homogenized in 1 ml sterile saline and 50 µl of this

suspension was inoculated onto OX, BM and on MC to which a 30 μg

ceftazidime tablet was placed (MCC). All plates were incubated at 35°C in

ambient air atmosphere for a full 24 h before reading.

All colonies developing on one of the two chromogenic media or within

a 20-mm inhibition zone diameter of the ceftazidime disk on MCC

(referred as isolates growing on the media MCC) were subcultured. Gram-

negative isolates were identified and tested for susceptibility using

Phoenix panels (Becton-Dickinson). The presence of an ESBL was

confirmed by double combination disc test using ceftazidime (30 µg) and

cefotaxime (30 µg) alone or in association with clavulanic acid (10 µg).

All Enterobacteriaceae isolates and non-fermenting Gram-negative

isolates showing resistance patterns compatible with acquired resistance

mechanisms to -lactam antimicrobials were referred to the bacteriology

laboratory of Mont-Godinne hospital for molecular characterization

including PCR detection of ESBLs, oxacillinases or carbapenemases.

An isolate was categorized as ESBL positive when the ESBL phenotype

displayed with the double disk test was confirmed by genotypic

characterization with the multiplex PCR.

BM OX

E. coli 45 45 45

K. pneumoniae 22 22 22

E. aerogenes 20 20 20

E. cloacae 5 5 5

C. freundii 3 3 3

K. oxytoca 3 3 3

Carbapenemase K. pneumoniae 2 2 2

E. coli 9 9 12

E. aerogenes 11 11 11

C. freundii 6 5 6

M. morganii 2 3 3

K. pneumoniae 2 2 2

K1-OXY penicillinase K. oxytoca 8 8 8

OXA-1/-30 penicillinase E. coli 3 3 6

E. coli 0 0 4

K. pneumoniae 0 0 2

E. aerogenes 0 0 1

K. oxytoca 0 0 1

P. aeruginosa 5 5 5

A. baumannii 1 1 1

P. aeruginosa 4 4 4

A. baumannii 4 4 4

P. aeruginosa 9 8 9

A. baumannii 2 2 2

Intrinsic resistance S. maltophilia 13 2 15

P. aeruginosa 3 3 3

A. baumannii 1 0 1

Enterobacteria

ESBL

AmpC cephalosporinase

Susceptible (Wild-type)

Number of

isolates

tested:Group Resistance mechanism Species

Number of isolates

growing on:

Non fermenters

ESBL

Acquired carbapenemase

Chromosomal cephalosporinase

and/or impermeability

Susceptible (Wild-type)

MCC BM OX All media

E. coli 36 (32) 52 (45) 54 (47) 62 (50)

E. cloacae 10 (5) 10 (4) 9 (4) 11 (5)

K. pneumoniae 7 (6) 8 (7) 7 (7) 9 (7)

E. aerogenes 7 (3) 7 (3) 6 (3) 7 (3)

K. oxytoca 0 5 3 5

M. morganii 3 (1) 2 (1) 5 (1) 5 (1)

C. freundii 3 (1) 3 (2) 3 (1) 4 (2)

S. marcescens 0 1 (1) 2 (2) 2 (2)

C. farmeri 0 2 2 2

H. alvei 0 2 0 2

P. aeruginosa 5 23 41 43

P. putida 0 6 9 9

S. maltophilia 2 9 0 9

A. baumannii 1 6 1 6

A. lwoffii 1 1 0 1

Yeast 2 0 0 2

E. faecium 0 2 0 2

E. faecalis 0 1 0 1

Species

Number of isolates growing on (ESBL-producing):

Non fermenters

and others

Enterobacteria

Group

TP (a) FP (b) FN

MCC 44 24 15 74,6% 96,8% 64,7% 96,8%

BM 56 59 3 94,9% 94,9% 48,7% 99,3%

OX 56 64 3 94,9% 95,1% 46,7% 99,3%

BM 51 21 8 86,4% 95,5% 70,8% 98,2%

OX 56 20 3 94,9% 95,7% 73,7% 99,3%Selected samples (c)

Samples considered Medium

Number of samples

PPV NPVSpecificity

All samples

Sensitivity

Interpretation of isolates on Brilliance ESBL® (OX)

Abbreviations: TP (True positive), FP (False positive), FN (False negative), PPV (Positive predictive value)

(a) A sample was assigned as TP when at least one ESBL positive isolate was recovered in the sample.

(b) A sample was assigned as FP when the isolate(s) recovered were not confirmed as ESBL producers.

(c) When selection criteria for work-out (oxidase negative and colored colonies) was applied.

Table 3: Sensitivity, specificity, PPV and NPV for the three tested media

Table 2: Distribution of organisms recovered from 528 clinical samples on the three selective mediaTable 1: Species and resistance mechanisms distribution of the challenge strains tested on the

chromogenic media

Chromogenic media definitely proved superior to MCC (p<0.01) with

a substantial number of ESBL-producing isolates (20 CTX-M types out of

22) being missed by MCC.

The new Brilliance™ ESBL agar proved valuable and showed equally

high performance as the ChromID™ ESBL agar for the detection of

ESBL-producing Enterobacteriaceae yielding an excellent NPV of 99.3%

at 24h and appeared as a very promising screening method which

could enable rapid exclusion of patients not carrying ESBL.

We confirmed the lack of ability of the two chromogenic media to

differentiate ESBL-mediated resistance from other closely resembling

resistance mechanisms phenotypes (e.g. AmpC, K1 penicillinase

overproduction, OXA-1/OXA-30 penicillinase). Confirmation of ESBL

production by further characterization tests for all colonies growing

on these chromogenic media is therefore required.

1. Paterson, D. L., and R. A. Bonomo. Clin Microbiol Rev 2005;18:657-86.

2. Lucet, J. C., D. Decre, A. Fichelle, M. L. Joly-Guillou, M. Pernet, C. Deblangy, M. J. Kosmann, and

B. Regnier. Clin Infect Dis 1999;29:1411-8.

3. Meyer, E., A. Serr, C. Schneider, S. Utzolino, W. V. Kern, R. Scholz, and M. Dettenkofer. Infect

Control Hosp Epidemiol 1999;30:103-5.

4. Glupczynski Y, Berhin C, Bauraing C, Bogaerts P. J Clin Microbiol. 2007; 45(2):501-5.

When considering only colored colonies of the Enterobacteriaceae

isolates, OX yielded a higher sensitivity than BM (94.9% vs 86.4% of the

59 ESBL positive samples), although the difference did not reach

statistical significance (p=0.1).

OX proved superior to BM for the

detection of C. freundii which always had

a green coloration on the former medium

while they were systematically colorless

on BM.

Large variability in the color of the

colonies displayed by E. coli on OX

which in some cases led to difficulties

in differentiating E. coli strains from

other species belonging to the KESC

group.

Regarding the non-fermenters, no

significant differences could be

observed between the two chromogenic

media except for the marked inhibitory

activity of OX against S. maltophilia

which grew on BM.

a) E. coli

b) E. coli lacking β-

galactosidase

c) Klebsiella,

Enterobacter,

Serratia and

Citrobacter

(KESC) group

d) Proteaceae group

e) f) P. aeruginosa

a c e

b d f

BM

BM

BM

OX

OX

OX

MONT-

GODINNE

MONT-

GODINNE