LETTER TO THE EDITOR

Blue-Carba, an Easy Biochemical Test for Detection of Diverse

Carbapenemase Producers Directly from Bacterial Cultures
J. Pires, Â. Novais, L. Peixe
REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

enem ␤-lactam ring through the acidification of a phenol red so-

Q uick, simple, and reliable methods are needed for laboratory
detection of carbapenemases that are widely disseminated
among Gram-negative bacteria, in order to improve the detection
lution used as color indicator. In the Blue-Carba test variant, bro-
mothymol blue was selected as the indicator, since it includes the
and surveillance of these clinically relevant bacteria in an epide- optimal pH range (6.0 to 7.6) for most ␤-lactamases (pH ⫽ 6.8),
miological context (1, 2). Recently, a highly sensitive and specific which was a key factor for a direct colony approach. A commer-
rapid biochemical test (Carba NP) was described to detect carbap- cially and widely available imipenem (Tienam 500; Merck Sharp &

Downloaded from http://jcm.asm.org/ on August 31, 2016 by guest

enemase production on Enterobacteriaceae and Pseudomonas spe- Dohme, France) was used as the substrate for carbapenemases.
cies extracts prepared with a commercial buffer (B-PER II) (3). The test solution consisted of an aqueous solution of bromothy-
Here, we propose a modified test (Blue-Carba) that was validated mol blue at 0.04% (Merck Millipore, Germany) adjusted to pH
for the detection of carbapenemase-producing strains directly 6.0, 0.1 mmol/liter ZnSO4, and 3 mg/ml of imipenem, with a final
from bacterial cultures. pH of 7.0. A negative-control solution (0.04% bromothymol blue
One hundred one previously characterized Enterobacteriaceae solution, pH 7.0) was prepared to control the influence of bacte-
(n ⫽ 44), Acinetobacter (n ⫽ 43), and Pseudomonas (n ⫽ 14) rial components or products in the pH of the solution. A loop
species strains producing Ambler class A, B, and D carbapen- (approximately 5 ␮l) of a pure bacterial culture recovered from
emases (KPC, IMP, NDM, VIM, SPM, and OXA) and 49 noncar- Mueller-Hinton agar (bioMérieux, France) was directly sus-
bapenemase producers (susceptible or nonsusceptible to carbap- pended in 100 ␮l of both test and negative-control solutions in a
enems) were tested (Table 1). Carbapenemase production was 96-well microtiter plate and incubated at 37°C with agitation (150
assessed by standard phenotypic tests, PCR and sequencing, rpm) for 2 h. Carbapenemase activity was revealed when the test
and/or spectrophotometric assays (2, 4). The MICs for carbapen- and negative-control solutions, respectively, were (i) yellow ver-
ems were determined using Etest (4). The Carba NP method relies sus blue, (ii) yellow versus green, or (iii) green versus blue. Non-
on the detection in a bacterial extract of hydrolysis of the carbap- carbapenemase producers remained blue or green on both solu-
tions (Fig. 1). The test was performed in triplicate for all isolates,
yielding reproducible results.
The Blue-Carba test detected all carbapenemase producers
(Table 1) with 100% sensitivity and 100% specificity. All noncar-
bapenemase producers (including extended-spectrum ␤-lacta-
mase- and/or AmpC-producing isolates), with or without altera-
tions in outer membrane permeability, gave negative results
(Table 1). Different times were required to observe a positive re-
sult for different carbapenemases types (e.g., KPC or MBL at the
first 30 min versus most OXA-type enzymes at 1 h 30 min to 2 h 00
min). Furthermore, a higher inoculum resulted in clearer color
changes for OXA types from Acinetobacter spp.
Blue-Carba was demonstrated to have specificity and sensitiv-
ity (100%) similar to those of Carba NP test, and it presents addi-
tional advantages, as follows: (i) increased protocol simplicity due
to the direct use of colonies (instead of bacterial extracts); (ii)
significantly reduced cost per reaction (over 200⫻), taking into
account the use of Tienam (ca. 10⫻ cheaper than an imipenem
monohydrate formula) and the dispensability of the extraction
buffer (B-PER II), which is used to obtain bacterial extracts; and
(iii) the validation of the test for the detection of OXA-type car-
bapenemases commonly identified in Acinetobacter spp.

FIG 1 Representative results of the Blue-Carba test obtained from carbapen- Published ahead of print 9 October 2013
emase producers (A, B, and C) and non-carbapenemase producers (D) with Address correspondence to Luísa Peixe, [email protected].
test solution (left) and negative control solutions (right). (A) NDM-1-produc- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
ing E. coli. (B) OXA-23-producing A. baumannii. (C) OXA-48-producing K.
doi:10.1128/JCM.01634-13
pneumoniae. (D) E. coli ATCC 25922. The images were taken after 2 hours of
incubation.

December 2013 Volume 51 Number 12 Journal of Clinical Microbiology p. 4281– 4283 jcm.asm.org 4281
Letter to the Editor

TABLE 1 Acquired-carbapenemase and noncarbapenemase-producing isolates tested

MIC (␮g/ml)b
Group of acquired Blue-Carba
␤-lactamasea Variant Species (no. of isolates) IPM MEM ERT result Reference or source
Carbapenemase producers
Class A
KPC KPC-2 Klebsiella pneumoniae (2) 8–16 8–16 4–16 ⫹ This study; Rafael Cantón

KPC-3 K. pneumoniae (7) 0.5–⬎8 0.5–⬎8 1–⬎8 ⫹ Rafael Cantón

Class B
IMP IMP-5 Acinetobacter baumannii (1) ⬎32 ⬎32 NA ⫹ 5

Acinetobacter bereziniae (1) ⬎32 ⬎32 NA ⫹ This study

NDM NDM-1 Escherichia coli (4) 6–64 16–⬎32 ⬎16 ⫹ Laurent Poirel; Katie Hopkins
and Neil Woodford

K. pnemuoniae (3) 16–64 32–⬎32 ⬎16 ⫹ Katie Hopkins and Neil

Woodford
VIM VIM-1 K. pneumoniae (13) 0.5–⬎32 0.063–1 1–32 ⫹ This study; Rafael Cantón

Downloaded from http://jcm.asm.org/ on August 31, 2016 by guest

VIM-2 K. pneumoniae (1) 1 0.25 0.25 ⫹ This study
Pseudomonas aeruginosa (12) 16–⬎32 1–⬎32 NA ⫹ This study; 6, 7
Pseudomonas pseudoalcaligenes (1) ⬎32 ⬎32 NA ⫹ 8
VIM-34 K. pneumoniae (2) 1 0.5 0.5 ⫹ This study
SPM SPM-1 P. aeruginosa (1) ⬎32 ⬎32 NA ⫹ Laurent Poirel
Class D
OXA OXA-23 A. baumannii (14) ⬎32 ⬎32 NA ⫹ Paolo Visca; 9, 10

OXA-40 A. baumannii (17) ⬎32 ⬎32 NA ⫹ 9

Acinetobacter haemolyticus (1) ⬎32 ⬎32 NA ⫹ 11
Acinetobacter baylyi (2)c ⬎32 ⬎32 NA ⫹ 11
OXA-48 K. pneumoniae (12) 0.5–⬎32 0.5–⬎32 1–⬎32 ⫹ Rafael Cantón; Laurent Poirel
OXA-58-like A. baumannii (6) 0.5–⬎32 1–8 NA ⫹ Paolo Visca; 9
OXA-72 A. baumannii (1) ⬎32 ⬎32 NA ⫹ Ivana Goic-Barisic

Noncarbapenemase producers
Class A
CTX-M CTX-M-1 E. coli (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study

CTX-M-2 E. coli (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study

CTX-M-9 E. coli (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
Salmonalla enterica (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ 12
CTX-M-14 E. coli (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
CTX-M-15 E. coli (2) ⱕ1 ⱕ1 ⱕ0.25 ⫺ 13
Klebsiella oxytoca (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
K. pneumoniae (2) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
CTX-M-15 ⫹ SHV-12 E. coli (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ 13
CTX-M-15 K. pneumoniae (4)d 0.125–8 0.25–8 0.03–32 ⫺ 14
CTX-M-32 E. coli (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
GES GES-1 K. pneumoniae (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
SHV SHV-2 K. pneumoniae (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study

SHV-12 E. coli (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study

K. pneumoniae (2) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
TEM TEM-10 K. pneumoniae (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study

Serratia marcescens (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study

TEM-24 K. pneumoniae (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
TEM-52 K. pneumoniae (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
TEM-199 Proteus mirabilis (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
Class C
AmpC DHA-1 K. pneumoniae (1)d 0.125 0.016 ⬎32 ⫺ This study
Class A ⫹ C SHV-12 ⫹ DHA-1 K. pneumoniae (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ This study
Class D
OXA OXA-1/-30 S. enterica (1) ⱕ1 ⱕ1 ⱕ0.25 ⫺ 15

A. baumannii (4) 0.5–⬎32 0.5–⬎32 NA ⫺ Paolo Visca; Jaroslav Hrabák;

Harald Seifert
E. coli (1) 0.06–0.25 0.008–0.06 0.004–0.015 ⫺ Reference strain ATCC 25922
E. coli (1) 2 0.5 4 ⫺ This study
Enterobacter aerogenes (1) 16 2 2 ⫺ This study
E. aerogenes (1)d 4 0.5 2 ⫺ 14
Enterobacter cloacae (3) 0.5–2 0.25 2–32 ⫺ This study
K. pneumoniae (2) 0.125 0.125–4 0.5–8 ⫺ This study
K. pneumoniae (2)d 1 2 16 ⫺ 14
P. aeruginosa (4) ⬎32 ⬎32 NA ⫺ This study
P. aeruginosa (1)d 0.25 0.25 NA ⫺ José Claudio Pérez-Díaz
a
␤-Lactamases conferring resistance to extended-spectrum ␤-lactams.
b
IPM, imipenem; MEM, meropenem; ERT, ertapenem; NA, not applicable.
c
Transformant strains.
d
Isolates have deficiency in membrane permeability.

4282 jcm.asm.org Journal of Clinical Microbiology

 Letter to the Editor

In conclusion, Blue-Carba is an easier and cheaper alternative 4. CLSI. 2011. Performance standards for antimicrobial susceptibility test-
to the Carba NP test, allowing the detection of carbapenemase ing; twentieth informational supplement. CLSI document M100-S21.
Clinical and Laboratory Standards Institute, Wayne, PA.
activity directly from bacterial cultures of Enterobacteriaceae, 5. Da Silva GJ, Correia M, Vital C, Ribeiro G, Sousa JC, Leitão R, Peixe
Pseudomonas, and Acinetobacter species. L, Duarte A. 2002. Molecular characterization of blaIMP-5, a new inte-
gron-borne metallo-␤-lactamase gene from an Acinetobacter baumannii
ACKNOWLEDGMENTS nosocomial isolate in Portugal. FEMS Microbiol. Lett. 215:33–39.
6. Cardoso O, Leitão R, Figueiredo A, Sousa JC, Duarte A, Peixe LV. 2002.
This work was supported by Fundação para a Ciência e Tecnologia Metallo-␤-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa
through grants no. PEst-C/EQB/LA0006/2011, PTDC/AAC-AMB/103386/ from Portugal. Microb. Drug Resist. 8:93–97.
2008, EXPL/DTP-EPI/0196/2012, and FCOMP-01-0124-FEDER-027745. 7. Quinteira S, Peixe L. 2006. Multiniche screening reveals the clinically
Â.N. was supported by a Marie Curie Intra European fellowship (PIEF- relevant metallo-␤-lactamase VIM-2 in Pseudomonas aeruginosa far from
GA-2009-255512). the hospital setting: an ongoing dispersion process? Appl. Environ. Micro-
We thank (in alphabetical order) Rafael Cantón (Servicio de Mi- biol. 72:3743–3745.
8. Quinteira S, Ferreira H, Peixe L. 2005. First isolation of blaVIM-2 in an
crobiología, Hospital Universitário Ramón y Cajal, Madrid, Spain),
environmental isolate of Pseudomonas pseudoalcaligenes. Antimicrob.
Ivana Goic-Barisic (Clinical Department of Microbiology and Parasi- Agents Chemother. 49:2140 –2141.
tology, Split University Hospital and School of Medicine, Split, Croa- 9. Grosso F, Quinteira S, Peixe L. 2011. Understanding the dynamics of
tia), Katie Hopkins (Antimicrobial Resistance and Healthcare-Associ- imipenem-resistant Acinetobacter baumannii lineages within Portugal.

Downloaded from http://jcm.asm.org/ on August 31, 2016 by guest

ated Infections Reference Unit, Public Health England, London, Clin. Microbiol. Infect. 17:1275–1279.
United Kingdom), Jaroslav Hrabák (Department of Microbiology, 10. Grosso F, Carvalho KR, Quinteira S, Ramos A, Carvalho-Assef APDA,
Faculty of Medicine and University Hospital in Plzeň, Charles Univer- Asensi MD, Peixe L. 2011. OXA-23-producing Acinetobacter baumannii:
sity in Prague, Plzeň, Czech Republic), José Claudio Pérez-Díaz (Ser- a new hotspot of diversity in Rio de Janeiro? J. Antimicrob. Chemother.
vicio de Microbiología, Hospital Universitário Ramón y Cajal, Ma- 66:62– 65.
11. Grosso F, Quinteira S, Poirel L, Novais Â, Peixe L. 2012. Role of
drid, Spain), Laurent Poirel (Service de Bactériologie-Virologie,
common blaOXA-24/OXA-40-carrying platforms and plasmids in the spread
Hôpital de Bicêtre, Paris, France), Harald Seifert (Institute for Medical of OXA-24/OXA-40 among Acinetobacter species clinical isolates. Antimi-
Microbiology, Immunology and Hygiene, University of Cologne, Co- crob. Agents Chemother. 56:3969 –3972.
logne, Germany), Paolo Visca (Department of Biology, Roma Tre Uni- 12. Antunes P, Mourão J, Alves T, Campos J, Novais C, Novais Â, Peixe L.
versity, Rome, Italy), and Neil Woodford (Antimicrobial Resistance 2013. Salmonella enterica serotype Bovismorbificans, a new host for CTX-
and Healthcare-Associated Infections Reference Unit, Public Health M-9. Int. J. Antimicrob. Agents. 41:91–93.
England, London, United Kingdom) for the gift of strains, in most 13. Novais Â, Pires J, Ferreira H, Costa L, Montenegro C, Vuotto C,
cases involving on-going or finished cooperation studies. Donelli G, Coque TM, Peixe L. 2012. Characterization of globally spread
Escherichia coli ST131 isolates (1991 to 2010). Antimicrob. Agents Che-
mother. 56:3973–3976.
REFERENCES 14. Novais Â, Rodrigues C, Branquinho R, Antunes P, Grosso F, Boaven-
1. Patel G, Bonomo R. 2013. “Stormy waters ahead”: global emergence of tura L, Ribeiro G, Peixe L. 2012. Spread of an OmpK36-modified ST15
carbapenemases. Front. Microbiol. 4:48. doi:10.3389/fmicb.2013.00048. Klebsiella pneumoniae variant during an outbreak involving multiple car-
2. Nordmann P, Poirel L. 2013. Strategies for identification of carbapen- bapenem-resistant Enterobacteriaceae species and clones. Eur. J. Clin. Mi-
emase-producing Enterobacteriaceae. J. Antimicrob. Chemother. 68:487– crobiol. Infect. Dis. 31:3057–3063.
489. 15. Antunes P, Machado J, Sousa JC, Peixe L. 2004. Dissemination amongst
3. Dortet L, Poirel L, Nordmann P. 2012. Rapid identification of carbap- humans and food products of animal origin of a Salmonella typhimurium
enemase types in Enterobacteriaceae and Pseudomonas spp. by using a clone expressing an integron-borne OXA-30 ␤-lactamase. J. Antimicrob.
biochemical test. Antimicrob. Agents Chemother. 56:6437– 6440. Chemother. 54:429 – 434.

December 2013 Volume 51 Number 12 jcm.asm.org 4283