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Detection of carbapenemase
production in Enterobacteriaceae
and Pseudomonas species by
carbapenemase Nordmann–Poirel test
Website:
www.jlponline.org Morubagal R. Rao, Pooja Chandrashaker, Rashmi P. Mahale, Sowmya G. Shivappa,
DOI:
Ranjitha S. Gowda, Vidyavathi B. Chitharagi
10.4103/JLP.JLP_132_18

Abstract:
PURPOSE: Multidrug‑resistant organisms causing community‑acquired and hospital‑acquired
infections are increasing at a dangerous rate. Carbapenemase‑producing Enterobacteriaceae and
Pseudomonas species are an important source of concern since these organisms are not only
resistant to beta‑lactam antibiotics but also show cross‑resistance to other groups of antibiotics.
In the present study, rapid detection of these carbapenemase‑producing Enterobacteriaceae and
Pseudomonas species by carbapenemase Nordmann–Poirel (Carba NP) test was evaluated by
comparing with modified Hodge test (MHT).
MATERIALS AND METHODS: Imipenem‑resistant Enterobacteriaceae and Pseudomonas species
isolated from various samples such as pus, blood, sputum, urine, and endotracheal aspirates were
processed for carbapenemase detection by MHT and Carba NP test. Kappa analysis was done to
evaluate the percentage agreement between the two tests.
RESULTS: Seventy imipenem‑resistant Enterobacteriaceae and Pseudomonas isolates were
analyzed in the present study for carbapenemase production. 63.41% of Enterobacteriaceae and
34.48% of Pseudomonas species were carbapenemase producers considering both the methods. By
MHT, 36 (51.42%) isolates and, by Carba NP test, 35 (50%) isolates were positive for carbapenemase
production out of the 70 isolates.
CONCLUSION: Carba NP test when compared to MHT is a simple, rapid, cost‑effective biochemical test
which can be used in all laboratories in the identification of life‑threatening carbapenemase‑producing
Gram‑negative bacteria.
Key words:
Carbapenemase, carbapenemase Nordmann–Poirel, Enterobacteriaceae, modified Hodge test,
Pseudomonas

Introduction cephalosporinase (AmpC)‑producing

Department of organisms are resistant to almost all

M
Microbiology, JSS Medical
College, JSS Academy
ultidrug‑resistant organisms β‑lactams with the exception of
of Higher Education causing community‑acquired carbapenems. Treatment of choice
and Research, Mysore, and hospital‑acquired infections are for these ESBL‑ or AmpC‑producing
Karnataka, India increasing at a dangerous rate globally, isolates is carbapenems.[2] Therefore, it is
especially among Enterobacteriaceae and important to preserve the clinical efficacy
Address for
correspondence: nonfermenters. [1] Extended‑spectrum of carbapenems (imipenem, ertapenem,
Dr. Ranjitha S. Gowda, β‑lactamase (ESBL)‑ and acquired meropenem, and doripenem). However,
Department of in recent times, there is an increase in
Microbiology, JSS Medical
College, Shivarathreswara This is an open access journal, and articles are
Nagar, Mysore ‑ 570 015, distributed under the terms of the Creative Commons How to cite this article: Rao MR, Chandrashaker P,
Karnataka, India. Attribution‑NonCommercial‑ShareAlike 4.0 License, which Mahale RP, Shivappa SG, Gowda RS, Chitharagi VB.
E‑mail: ranju.vish85@ allows others to remix, tweak, and build upon the work Detection of carbapenemase production in
gmail.com non‑commercially, as long as appropriate credit is given and Enterobacteriaceae and Pseudomonas species
the new creations are licensed under the identical terms. by carbapenemase Nordmann–Poirel test. J Lab
Submission: 07‑10‑2018 Physicians 2019;11:107-10.
Accepted: 03-04-2019 For reprints contact: reprints@medknow.com

© 2019 Journal of Laboratory Physicians | Published by Wolters Kluwer - Medknow 107

 Rao, et al.: Carbapenemase detection by Carba NP test

reporting of carbapenem‑resistant Enterobacteriaceae and Pseudomonas species were further processed for
worldwide, probably as a result of carbapenemase carbapenemase production by MHT and Carba NP test.
gene acquisition[3] resulting in treatment failure due to Kappa analysis was done to evaluate the percentage
carbapenem usage. Various mechanisms of carbapenem agreement between MHT and Carba NP tests.
resistance in Enterobacteriaceae are due to a decrease in
bacterial outer‑membrane permeability, with excess Modified Hodge test
production of β‑lactamases with no carbapenemase Lawn culture of ATCC Escherichia coli 25922 at a
activity or expression of carbapenemases.[4,5] Klebsiella turbidity equivalent to that of 0.5 McFarland was made
pneumoniae carbapenemase (Ambler Class A); Verona onto the Mueller‑Hinton agar plate. After drying, an
integron–encoded metallo‑β‑lactamase, imipenemase, imipenem (10 µg) disc was placed at the center of the
and New Delhi metallo‑β‑lactamase (all Ambler Class B); plate. The test strain and control strains (a known
and oxacillinase‑48 (Ambler class D) are some examples carbapenemase‑producing Pseudomonas aeruginosa was
of carbapenemases reported in Enterobacteriaceae.[6] used as positive control, and E. coli ATCC 25922 was
used as negative control) were heavily streaked from
Due to other resistance mechanisms, most of the the edge of the imipenem disc to the periphery of the
times carbapenemase‑producing Enterobacteriaceae plate in different directions. The plates were incubated
and Pseudomonas species show resistance to other at 37°C for 18–24 h.
groups of drugs also leading to multidrug‑resistant
or pandrug‑resistant isolates. Rampant spread of such Interpretation
isolates is an important source of concern globally.[7] This The presence of a cloverleaf type of zone of inhibition
shows that for the selection of appropriate therapeutic near the test/positive control organism was interpreted
schemes and the implementation of infection control as MHT positive.[12]
measures, detection of carbapenemase producers is
important.[8,9] Rapid identification of carbapenemase Carbapenemase Nordmann–Poirel test
is the need of today’s clinical practice. Ultraviolet The Carba NP test for Enterobacteriaceae and Pseudomonas
spectrophotometry, matrix‑assisted laser desorption spp. was performed as follows:
ionization–time of flight technique, and molecular
methods are few examples of techniques available Two 1.5‑ml low‑bind protein microcentrifuge
for rapid detection. Even though these methods have tubes (Eppendorf), each containing 100 µl of a 20‑mM
good sensitivity and specificity, they require trained Tris‑HCL lysis buffer, were individually inoculated
microbiologists and expensive equipment. Molecular with a 1‑µl loopful of bacterial colony (18–24 h old, loop
methods which are considered as gold standard swept through pure culture), and bacterial suspensions
method may fail to detect unknown carbapenemase were vortexed for 5 min. To the first tube, 100 µl of
genes not included in gene panel.[10] To overcome all 0.5% (wt/vol) phenol red solution with 10‑mM zinc
these drawbacks, a biochemical test (carbapenemase sulfate (solution A, buffered to pH 7.8 by adding
Nordmann–Poirel [Carba NP] test) based on a technique 0.1 N NaOH) was then added, and the tube was vortexed.
designed to identify the hydrolysis of the β‑lactam To the second tube, 100 µl of solution A with imipenem
ring of a carbapenem has been developed. [11] The dissolved directly in solution A to a final concentration
present study was undertaken to evaluate Carba NP of 6 mg/ml was added and then vortexed. A mixture of
test in discriminating carbapenemase producers from the phenol red solution and the enzymatic suspension
nonproducers by comparing with modified Hodge being tested was incubated at 37°C for a maximum of
test (MHT). 2 h.[13] A known carbapenemase‑producing P. aeruginosa
was used as positive control, and E. coli ATCC 25922 was
Materials and Methods used as negative control.

This study was conducted in the department of After incubation, the presence of any carbapenemase,
microbiology of a teaching hospital. Ethical clearance which hydrolyzes imipenem into its carboxylic form,
certificate was obtained from the Institutional Ethical leading to a pH decrease, was detected by a color change
Committee. Various samples such as pus, blood, of phenol red solution (red to yellow/orange), while
sputum, urine, and endotracheal aspirates received in tubes remain red in the absence of carbapenemase.
the laboratory were inoculated on a sterility‑checked
MacConkey agar and blood agar plates and incubated Interpretation
at 37°C for 18–24 h. Based on the growth on MacConkey 1. F i r s t t u b e a n d s e c o n d t u b e r e m a i n i n g
agar and blood agar, isolates were further processed in red – noncarbapenemase‑producing isolate
VITEK 2 systems, for identification and antimicrobial 2. First tube remaining red and second tube turning
susceptibility. Imipenem‑resistant Enterobacteriaceae yellow/orange – carbapenemase‑producing isolate.

108 Journal of Laboratory Physicians - Volume 11, Issue 2, April-June 2019

 Rao, et al.: Carbapenemase detection by Carba NP test

Results revealed that the strength of agreement between the two

tests is considered very good (kappa = 97%, confidence
Seventy imipenem‑resistant Enterobacteriaceae interval = 0.916–1.00).
(19 – Klebsiella species, 15 – E. coli, 3 – Enterobacter
cloacae, 2 – Morganella morganii, and 2 – Serratia species) Discussion
and Pseudomonas species (29) isolates were analyzed in
the present study. One of the greatest advances of modern medicine is
the development of antibiotics for the treatment of
Out of 29 Pseudomonas species, 14 were from pus samples infectious disease. Unfortunately, effectiveness of many
followed by 12 from endotracheal aspirates. Out 19 antimicrobial agents is under threat due to the emergence
Klebsiella species, 8 were from pus and 7 from urine. of antibiotic resistance among bacteria. In order to
Sample‑wise distribution of isolates is shown in Table 1. control the emergence of drug resistance, the irrational
use of antibiotics should be controlled. One of the ways
Out of 29 imipenem‑resistant Pseudomonas species of controlling antibiotic misuse is rapid detection and
studied, 17 (58.6%) isolates were resistant to meropenem reporting of drug resistance mechanisms in clinical
and 18 (62.0%) isolates were resistant to doripenem. isolates, which helps in the selection of appropriate
Twenty‑seven (93.1%) isolates were found to be sensitive antibiotics for treatment. Keeping this in mind, the
to colistin. present study evaluated Carba NP test for rapid
detection of carbapenemase among Enterobacteriaceae
Of the 19 Klebsiella species studied, total resistance
and Pseudomonas species.
was observed to imipenem and meropenem. Sixteen
(84.2%) isolates were found resistant to ertapenem. A total of 70 clinical isolates (29 Pseudomonas and 41
Thirteen (68.4%) isolates were resistant to tigecycline Enterobacteriaceae) were analyzed for carbapenemase
and 15 (78.94%) isolates were sensitive to colistin. Of production. 63.41% of Enterobacteriaceae were
the 15 E. coli studied, total resistance was observed to carbapenemase producers (66.66% of E. coli and 73.68%
imipenem and meropenem. Ten (71.4%) were sensitive of Klebsiella species). Chauhan et al.[14] reported 87.01% of
to tigecycline and 13 (92.8%) isolates were found to be E. coli and 91.51% of Klebsiella spp. as carbapenemase
sensitive to colistin. producers by MHT. These findings suggest that
carbapenemase production is on rise in Enterobacteriaceae,
Out of 70 isolates analyzed for carbapenemase
particularly in Klebsiella species. 34.48% Pseudomonas
production, 36 (51.42%) isolates were carbapenemase
species were detected as carbapenemase producers,
producers by MHT and 35 (50%) isolates were detected
and similar findings were observed in a study done
as carbapenemase producers by Carba NP test. Out
by ElMasry et al., [15] in which 37% of Pseudomonas
of 29 pseudomonas species, 10 (34.48%) isolates were
species were reported as carbapenemase producers
detected as carbapenemase producer by MHT. Out of
by  polymerase chain reaction. However, by MHT,
these 10 isolates, one isolate gave negative result for
carbapenemase production by Carba NP test. positivity was increased to 48.1%.

Among 41 Enterobacteriaceae, 26 (63.41%) isolates One Pseudomonas aeruginosa strain in the present
were detected as carbapenemase producers. Out of study was positive for carbapenemase production by
19 Klebsiella species, 14 (73.68%) and, out of 15 E. coli, MHT but negative by Carba NP test. Several studies
10 (66.66%) were detected as carbapenemase producers have shown that  GES‑type carbapenemase‑producing
by both MHT and Carba NP test, respectively. One Pseudomonas species may not be detected by Carba NP
Morganella out of 2 and 1 Enterobacter species out of 3 test.[16] This could be one of the reasons in the present
were carbapenemase producers by both the methods. study for Carba NP test showing negative results.
Both Serratia species were negative for carbapenemase However, in this study, molecular analysis was not
production by both the methods. Kappa analysis performed to comment on sensitivity or specificity of
Carba NP test.
Table 1: Sample‑wise distribution of clinical isolates
Organism Pus Endotracheal Urine Blood Sputum In the present study, MHT test was performed using
aspirates imipenem disc instead of ertapenem or meropenem
Pseudomonas species (29) 14 12 0 0 3 disc because we were able to reproduce and interpret
Klebsiella species (19) 5 10 2 2 0 results better using imipenem disc than meropenem.
Escherichia coli (15) 8 0 7 0 0 Even though both Carba NP and MHT detected
Enterobacter cloacae (3) 2 0 0 1 0 carbapenemase producers almost equally except for one
Serratia marcescens (2) 0 2 0 0 0 strain which gave negative results with Carba NP test,
Morganella morganii (2) 2 0 0 0 0 MHT at times was difficult to interpret.
Journal of Laboratory Physicians - Volume 11, Issue 2, April-June 2019 109
 Rao, et al.: Carbapenemase detection by Carba NP test

Conclusion 6. Nordmann P, Naas T, Poirel L. Global spread of

carbapenemase‑producing Enterobacteriaceae. Emerg Infect Dis
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