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Objectives: To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important carbapenemase and minor extended-spectrum β-lactamase (ESBL) resistance genes, according to the international ISO 15189 particular requirements for the quality and competence of medical laboratories. Methods: Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced isolates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system, Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by the Belgian organization for accreditation (BELAC). Results: Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by BELAC, and the PCR protocols were accepted as accredited according to ISO 15189. Conclusions: Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards. This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.
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Validation of carbapenemase and extended-spectrum b-lactamase
multiplex endpoint PCR assays according to ISO 15189
P. Bogaerts1*, R. Rezende de Castro1, R. de Mendonc¸a2, T-D. Huang1, O. Denis2and Y. Glupczynski1
1
Laboratory of Bacteriology, CHU UCL Mont-Godinne-Dinant, Yvoir, Belgium;
2
Laboratory of Bacteriology, ULB– Erasme Hospital,
Brussels, Belgium
*Corresponding author. Tel: +32-81-42-32-41; Fax: +32-81-42-32-04; E-mail: pierre.bogaerts@uclouvain.be
Received 2 January 2013; returned 26 January 2013; revised 29 January 2013; accepted 31 January 2013
Objectives: To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important
carbapenemase and minor extended-spectrum b-lactamase (ESBL) resistance genes, according to the inter-
national ISO 15189 particular requirements for the quality and competence of medical laboratories.
Methods: Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed
internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and
accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced iso-
lates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to
PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system,
Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by
the Belgian organization for accreditation (BELAC).
Results: Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three
EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by
BELAC, and the PCR protocols were accepted as accredited according to ISO 15189.
Conclusions: Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A
ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards.
This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.
Keywords: accreditation, minor ESBLs, molecular detection
Introduction
The worldwide spread of genes conferring resistance to broad
spectrum b-lactams including carbapenems in Gram-negative
bacteria is a source of global concern.
13
Class A extended-
spectrum b-lactamase (ESBL)-encoding genes such as bla
TEM
,
bla
SHV
and especially bla
CTX-M
have largely disseminated world-
wide among Enterobacteriaceae. Other bla genes encoding
minor ESBLs (bla
BEL,
bla
VEB,
bla
GES and
bla
PER
) are more rarely
reported, although they have also been observed worldwide
especially among Gram-negative non-fermenters.
4
Even more worrying is the recent emergence and spread of
genes encoding carbapenemases.
3
Although class D carbape-
nem-hydrolysing b-lactamases of group OXA-23, OXA-24,
OXA-58 or OXA-143 type are almost exclusively reported in Aci-
netobacter baumannii,
5
OXA-48 (and related types) seems to
be exclusively and widely expressed in Enterobacteriaceae.
6
Besides OXA-48, the spread of carbapenemases of class A (KPC
and some GES variants such as GES-2 and GES-5) and of class
B (VIM, IMP and NDM) have been reported, albeit at differing fre-
quencies, in Enterobacteriaceae, Pseudomonas aeruginosa and
Acinetobacter spp.
1,7,8
Rapid and reliable detection methods are important for the
early implementation of infection control measures and for
preventing the subsequent dissemination of ESBLs and of carba-
penemases. Although a promising, rapid and easy detection
method based on the antibiotic hydrolytic properties of the
expressed b-lactamases has been recently published,
9,10
con-
firmation and identification of the precise types of b-lactamase
genes involved still need the use of molecular PCR-based
methods.
There is a plethora of PCR methods described in the literature
that are often validated only internally.
11 15
Nevertheless,
quality standards requirements for medical laboratories are
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now evolving as a general rule, and compliance with the inter-
national ISO 15189 requirements for the quality and compe-
tence of medical laboratories is becoming mandatory.
16
The
accreditation process according to ISO 15189 represents a
highly challenging task for medical laboratories. One particular
difficulty is related to the fact that these requirements have to
be fulfilled, but there are no universal ways or hints about how
to reach them.
We present here the validation scheme for three multiplex
endpoint PCR assays targeting some of the most epidemiologi-
cally relevant carbapenemases (PCR CARBA targeting bla
VIM,
bla
IMP,
bla
NDM,
bla
KPC
and bla
OXA-48
and PCR OXACARBA targeting
bla
OXA-23,
bla
OXA-24
and bla
OXA-58
) and the minor ESBLs (PCR
MINESBL targeting bla
BEL,
bla
GES,
bla
PER
and bla
VEB
). The latter
two tests were particularly important for our activity of reference
centre as no commercial assay is currently available for these
targets. These PCR assays were validated and accredited accord-
ing to the international ISO 15189 standards after an external
audit performed by the Belgian accreditation body BELAC. This
validation scheme could be useful for implementing accredited
home-made PCRs in the routine laboratory.
Materials and methods
Bacterial isolates (Table 1)
A collection of 137 Gram-negative clinical isolates obtained from the
European FP7 TEMPOtest-QC consortium Number 241742 (http://www.
tempotest-qc.eu/newweb/index.php?pageId=12) was used to validate
the three multiplex PCR assays (82 isolates for PCR CARBA, 79 for PCR
MINESBL and 68 for PCR OXACARBA). This collection comprised 56 Enter-
obacteriaceae isolates [Klebsiella pneumoniae(n¼21), Escherichia coli
(n¼8), Citrobacter spp. (n¼8), Enterobacter spp. (n¼6), Klebsiella
oxytoca (n¼3), Serratia marcescens (n¼2), Providencia spp. (n¼2),
Proteus spp.(n¼2), Hafnia alvei (n¼1), Morganella morganii (n¼1), Sal-
monella enterica (n¼1) and Aeromonas hydrophila (n¼1)] and 81 Gram-
negative non-fermenters [Pseudomonas spp. (n¼41), Acinetobacter spp.
(n¼39) and Alcaligenes xylosoxidans (n¼1)] expressing various resist-
ance genes.
Design of primers (Table 2)
For each gene family, all alleles referenced on the Lahey Clinic web site
(http://www.lahey.org/Studies/) were uploaded from GenBank databases
and aligned using the ClustalX software version 2.0. Primers were
designed within the common coding region of the published alleles. An
additional primer pair that targets the chromosomal AmpC of A. bauman-
nii (bla
ADC
) was used as an internal PCR/extraction control. All the primers
apart from the forward primer targeting bla
VEB17
were designed in our
laboratory.
DNA extraction and multiplex PCR assays
A single colony was suspended in 200 mL of distilled water, and 10 mLof
a McFarland 3 turbidity standard of A. baumannii ATCC 19606 was added
to the suspension before extraction as an internal extraction control
(958C for 10 min in a dry bath). The 25 mL amplification mixture con-
tained 2 mL of DNA extract, 12.5 mLof2×master mix multiplex PCR Kit
(Qiagen Benelux, Antwerp, Belgium) and 200 mM of each primer
(except for IMP, for which the concentration of the primers was raised
to 600 mM). PCR was performed on a ABI 2720 thermocycler (Life Tech-
nologies Europe BV, Gent, Belgium) under the following conditions:
15 min at 958C and 30 cycles of 30 s denaturation at 948C; 90 s anneal-
ing at 578C and 90 s elongation at 728C; and a final elongation step at
728C for 10 min.
Each PCR run had to include three PCR controls: a positive resistance
gene control including each of the targeted genes, a DNA extraction posi-
tive control including only the internal extraction control suspended in
water, and a negative control (only water, no DNA). The amplicons
were visualized by capillary electrophoresis on a QIAxcel instrument
(Qiagen Benelux) using the QIAxcel high-resolution kit, QX DNA size
marker 100– 2500 bp and QX alignment markers 15/5000 bp according
to the manufacturer’s recommendations. The whole process, including
extraction and electrophoresis takes ,4 h. A negative result could only
be technically validated when the band corresponding to the internal ex-
traction control was present. In case of a positive result for any of the tar-
geted genes, the presence of the internal control does not need to be
taken into account.
Validation process
The validation process was based on the procedure proposed by Rabenau
et al.
18
for home-made qualitative nucleic acid testing. This process
includes the control of the specificity, sensitivity, reproducibility and ac-
curacy of each PCR multiplex assay. For sensitivity, at least 10 different
isolates positive for the gene to be detected were tested once. In cases
where the minimum number of isolates harbouring the targeted genes
was not available in the library, the available isolates were extracted
twice or more so as to reach 10 different sample preparations. For speci-
ficity, at least 20 isolates known to be negative for the targeted genes,
but possibly expressing other resistance genes representative of the
current b-lactamase epidemiology, were tested once. For reproducibility,
one isolate positive for each resistance gene was tested three times
intra-run and three times inter-run. Finally, accuracy was certified by
testing a panel prepared and sent by an external laboratory [external
quality control (EQC) process] comprising three positive isolates for
each target to be tested and three negative ones. This panel equally
comprised referenced isolates from the TEMPOtest-QC collection.
Results and discussion
The three multiplex PCR assays were validated with a panel of
137 characterized and referenced Gram-negative clinical isolates
(Table 1) according to the validation protocol presented in
the ‘Materials and methods’ section. This collection includes
30 metallo-b-lactamase-expressing isolates (10 VIM-, 10 IMP-
and 10 NDM-expressing isolates), 10 OXA-48-expressing isolates,
10 KPC-expressing isolates, 31 OXA-23, OXA-24 or OXA-58-
expressing isolates, 41 minor ESBL-expressing isolates (BEL,
VEB, GES and PER), 27 TEM-expressing isolates, 26 SHV-
expressing isolates and 14 CTX-M-expressing isolates. In add-
ition, 33 isolates expressing additional b-lactamases not
targeted by the PCR assays were used for specificity testing.
For each multiplex PCR assay, the presence of an extraction
control ruled out false-negative results due to PCR inhibition or
extraction faults. In the positive resistance gene control, the
presence of each band at the expected size confirms the ability
of the PCR to detect up to five resistance genes in a single PCR
(Figure 1). The positive extraction PCR control has to be per-
formed in a separate well as the corresponding 1059 bp ampli-
con tends to disappear in the presence of another resistance
gene. It highlights that the extraction/inhibition control does
not interfere with the detection of the targeted resistance
genes generating smaller amplicons.
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Table 1. Collection of characterized clinical isolates used for multiplex PCR assay validation
Species Number
bla
VIM,
bla
IMP
or bla
NDM
bla
OXA-48
bla
KPC
bla
OXA-23,
bla
OXA-24
or
bla
OXA-58 groups
bla
BEL,
bla
GES,
bla
PER or
bla
VEB
bla
TEMa
bla
SHVa
bla
CTX-M
of G1,
2 and 9
a
Other
b
-lactamases
b
Klebsiella pneumoniae 21 5 4 10 11 20 3 10
Escherichia coli 82 2 62 3 9
Enterobacter cloacae 42 2 13 4 1
Klebsiella oxytoca 31 1 1
Citrobacter freundii 31 11
Serratia marcescens 21 11
Enterobacter aerogenes 111
Enterobacter asburiae 1
Other Enterobacteriaceae
c
14 4 3 4 3 2
Acinetobacter baumannii 34 2 27 10 1 1
Acinetobacter spp. other than
A. baumannii
51 4 1
Pseudomonas aeruginosa 39 10 26 10
Pseudomonas spp. other than
P. aeruginosa
22
Total 137 30 10 10 31 41 27 26 14 33
a
Including both ESBLs and non-ESBLs.
b
Including plasmidic AmpC, carbenicillinases, oxacillinases and SPM metallo-b-lactamase.
c
Including Proteus vulgaris,Aeromonas hydrophila,Citrobacter spp., Hafnia alvei,Morganella morganii and Providencia spp.
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Table 2. Primer sequences and amplicon sizes
PCR name Targeted gene Primer sequence (5to 3)
a
Amplicon size
CARBA bla
NDM
Forward ACT TGG CCT TGC TGT CCT T 603 bp
Reverse CAT TAG CCG CTG CAT TGA T
bla
VIM
Forward TGT CCG TGA TGG TGA TGA GT 437 bp
Reverse ATT CAG CCA GAT CGG CAT C
bla
IMP
Forward ACA YGG YTT RGT DGT KCT TG 387 bp
Reverse GGT TTA AYA AAR CAA CCA CC
bla
KPC
Forward TCG CCG TCT AGT TCT GCT GTC TTG 353 bp
Reverse ACA GCT CCG CCA CCG TCA T
bla
OXA-48
Forward ATG CGT GTA TTA GCC TTA TCG 265 bp
Reverse CAT CCT TAA CCA CGC CCA AAT C
OXACARBA bla
OXA-23 group
Forward CCC CGA GTC AGA TTG TTC AAG G 330 bp
Reverse TAC GTC GCG CAA GTT CCT GA
bla
OXA-24/143 group
Forward GCA GAA AGA AGT AAA RCG GGT 271 bp
Reverse CCA ACC WGT CAA CCA ACC TA
bla
OXA-58 group
Forward GGG GCT TGT GCT GAG CAT AGT 688 bp
Reverse CCA CTT GCC CAT CTG CCT TT
MINESBL bla
PER
Forward AGT GTG GGG GCC TGA CGA T 725 bp
Reverse GCA ACC TGC GCA ATR ATA GCT T
bla
GES
Forward CTG GCA GGG ATC GCT CAC TC 600 bp
Reverse TTC CGA TCA GCC ACC TCT CA
bla
BEL
Forward CGA CAA TGC CGC AGC TAA CC 448 bp
Reverse CAG AAG CAA TTA ATA ACG CCC
bla
VEB
Forward CGA CTT CCA TTT CCC GAT GC 376 bp
Reverse TGT TGG GGT TGC CCA ATT TT
Inhibition control bla
ADCb
Forward GTA CCT CAA TTT ATG CGG RCA ATA C 1059 bp
Reverse TGC GYT CTT CAT TTG GAA TAC G
a
For degenerate primers: D ¼A, G or T; R ¼AorG;Y¼CorT;K¼GorT;W¼AorT.
b
ADC, Acinetobacter-derived cephalosporinase used as an inhibition/extraction control in each of the three multiplex PCRs.
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Sensitivity, specificity and reproducibility (including the extrac-
tion step) for each of the targeted genes were 100% (data not
shown). The resistance genes of the isolates included in the
three EQC panels were correctly identified by each PCR assay
(accuracy 100%; Figure 1). Regarding specificity, non-specific
amplifications generating fragments of unexpected sizes were
C+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 61 17 IC C– C+ 18 MW (a)
(b)
(c)
1 2 3 4 5 6 7 8 9 10 11 12 C– IC C +
MW
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 C– C+
ICMW
blaVEB
blaOXA–48
blaKPC
blaIMP
blaVIM
blaNDM
blaADC: IC
blaADC: IC
blaOXA-58
blaOXA-23
blaOXA-24
blaADC: IC
blaPER
blaGES
blaBEL
5000.0
2500.0
1500.0
1000.0
700.0
500.0
300.0
2500.0
1500.0
1000.0
700.0
500.0
300.0
100.0
100.0
2000.0
1200.0
800.0
600.0
400.0
5000.0
5000.0
2000.0
1200.0
800.0
600.0
400.0
200.0
15.0
2500.0
1500.0
1000.0
700.0
500.0
300.0
100.0
2000.0
1200.0
800.0
600.0
400.0
200.0
15.0
200.0
15.0
Figure 1. Capillary electrophoresis of amplicons obtained for accuracy testing (EQC ring test) of the three multiplex PCR assays. MW, molecular weight
(bp); IC, internal control, corresponding to bla
ADC
of Acinetobacter baumannii;C+, positive control consisting of a nucleic acid extracted from a mixture
of strains expressing the resistance genes to be targeted; C-, negative control (water only). (a) PCR CARBA targeting bla
VIM,
bla
IMP,
bla
NDM,
bla
KPC
and
bla
OXA-48
: lanes 1, 6 and 7, negative strains (Acinetobacter radioresistens OXA-23, A. baumannii PER-1 and Pseudomonas aeruginosa BEL-1); lanes 2, 8
and 9, KPC-producing strains (Klebsiella pneumoniae KPC-2); lanes 3, 5 and 12, VIM-producing strains (Citrobacter braakii VIM-1, P. aeruginosa VIM-4
and P. aeruginosa VIM-2); lanes 4, 14 and 16, OXA-48-producing strains (Enterobacter cloacae OXA-48, K. pneumoniae OXA-48 and E. coli OXA-48);
lanes 10, 11 and 15, IMP-producing strains (P. aeruginosa IMP-13, P. aeruginosa IMP-13 and P. aeruginosa IMP-7); and lanes 13, 17 and 18,
NDM-producing strains (K. pneumoniae NDM-1). (b) PCR OXACARBA targeting bla
OXA-23,
bla
OXA-24
and bla
OXA-58
: lanes 1, 2 and 6, negative strains
(Citrobacter freundii TEM-1, Proteus mirabilis CTX-M-2 and P. aeruginosa SPM); lanes 3, 7 and 8, OXA-23-producing strains (A. radioresistens OXA-23
and A. baumannii OXA-23); lanes 4, 9 and 11, OXA-24-like producing strains (A. baumannii OXA-72); and lanes 5, 10 and 12, OXA-58-producing
strains (Acinetobacter haemolyticus OXA-58, A. baumannii OXA-58 and Acinetobacter pittii OXA-58). (c) PCR MINESBL targeting bla
BEL,
bla
GES,
bla
PER
and bla
VEB
: lanes 1, 8 and 11, negative strains (C. braakii VIM-1, P. aeruginosa VIM-2 and P. aeruginosa IMP-7); lanes 2, 5 and 6, PER-producing
strains (A. baumannii PER-1); lanes 3, 4 and 13: BEL-producing strains (P. aeruginosa BEL-1); lanes 7, 9 and 10: GES-producing strains
(A. baumannii GES-12, P. aeruginosa GES-1 and P. aeruginosa GES-18); and lanes 12, 14 and 15, VEB-producing strains (P. aeruginosa VEB-1b, 1a, 1).
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observed in about 2% of the cases, and have to be interpreted as
negative results (Figure 1b, lane 1). These non-specific results
were often related to an Enterobacter asburiae isolate (data
not shown). No explanation could be found to explain this phe-
nomenon, although it was less frequently observed when the
primers were HPLC purified and was not observed when the
PCR was performed in simplex for each individual target. The val-
idation files were controlled by BELAC during an external audit
held in UCL Mont-Godinne on 19 March 2012, and the three
PCR protocols were accepted for accreditation according to ISO
15189 in July 2012 (BELAC Certificate 431-MED).
It is important to point out that, according to ISO 15189, an
efficient separation between the different work areas must be
provided in order to efficiently avoid cross-contamination. All
the processes described here only represent the analytical part
of the accreditation process. Pre-analytical and post-analytical
stages must also follow ISO 15189 standards, but these are
more difficult to export from one laboratory to another. Many ex-
cellent home-made PCRs (endpoint or real-time) have already
been published in the literature (e.g. Dallenne et al.,
11
Huang
et al.,
12
Naas et al.,
13
Poirel et al.,
14
Swayne et al.
15
and Naas
et al.,
19
although many other publications exist). Regarding
other multiplex PCRs for carbapenemases, each method presents
its own characteristics. For example, Huang et al.
12
proposed a
real-time TaqMan multiplex targeting different class D carbape-
nemases from A. baumannii, while Swayne et al.
15
published a
TaqMan PCR targeting five class A and D carbapenemases
encountered in Enterobacteriaceae. The three multiplex PCR
assays presented by Poirel et al.
14
are endpoint PCRs targeting
the largest panel of carbapenemase genes (11 targets). The im-
plementation of PCR in a laboratory will depend on its particular
needs, local epidemiology, technical resources and quality
requirements. Most methods are indeed efficient and have
already been peer-reviewed, but they would most probably not
be accepted as such by external auditors as conforming to ISO
15189. Major non-conformities with ISO 15189 standards are
the lack of an internal control able to rule out false-negative
results and the absence of accuracy testing by participating in
at least one annual EQC.
Testing accuracy is optimally achieved by participation in ex-
ternal quality assurance schemes organized by independent
bodies such as Quality Control for Molecular Diagnostics or the
United Kingdom National External Quality Assessment Service.
Unfortunately, these bodies do not yet organize EQC evaluations
for the detection of resistance genes. In such situations, ISO
15189 accepts that laboratories organize a so-called ring test
(a blind exchange of an EQC proficiency panel) to evaluate
the accuracy of their own methods. This is what was
performed with the strains obtained from the TEMPOtest-QC
consortium.
In summary, we report here the successful accreditation
process of three home-made multiplex PCR assays according
to the ISO 15189 standards. We believe that this validation
scheme should constitute a valuable tool for laboratories in
order to accredit their own protocols. Moreover, as these three
multiplex PCR assays are already accredited, they could easily
be implemented in other diagnostic laboratories through a
lighter verification procedure.
18
Funding
This work was supported by EU grant FP7-HEALTH-2009-SINGLESTAGE
TEMPOtest-QC, project 241742 and by INAMI/RIZIV funding of the
Belgian reference centres.
Transparency declarations
None to declare.
References
1Bush K, Fisher JF. Epidemiological expansion, structural studies, and
clinical challenges of new b-lactamases from Gram-negative bacteria.
Annu Rev Microbiol 2011; 65: 455– 78.
2Canton R, Lumb J. Emerging resistance in Gram-negative pathogens
and implications for clinical practice. Future Microbiol 2010; 6: 19– 22.
3Nordmann P, Cornaglia G. Carbapenemase-producing Enterobacte-
riaceae: a call for action! Clin Microbiol Infect 2012; 18: 411 –2.
4Naas T, Poirel L, Nordmann P. Minor extended-spectrum b-lactamases.
Clin Microbiol Infect 2008; 14 Suppl 1: 42–52.
5Poirel L, Bonnin RA, Nordmann P. Genetic basis of antibiotic resistance
in pathogenic Acinetobacter species. IUBMB Life 2011; 63: 1061– 7.
6Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the
phantom menace. J Antimicrob Chemother 2012; 67: 1597– 606.
7Cuzon G, Naas T, Truong H et al. Worldwide diversity of Klebsiella
pneumoniae that produce b-lactamase bla
KPC-
gene.Emerg Infect Dis
2010; 16: 1349– 56.
8Queenan AM, Bush K. Carbapenemases: the versatile b-lactamases.
Clin Microbiol Rev 2007; 20: 440– 58.
9Dortet L, Poirel L, Nordmann P. Rapid Identification of carbapenemase
types in Enterobacteriaceae and Pseudomonas spp. by using a
biochemical test. Antimicrob Agents Chemother 2012; 56: 6437– 0.
10 Nordmann P, Dortet L, Poirel L. Rapid detection of extended-
spectrum-b-lactamase-producing Enterobacteriaceae. J Clin Microbiol
2012; 50: 3016– 22.
11 Dallenne C, Da Costa A, Decre D et al. Development of a set of
multiplex PCR assays for the detection of genes encoding important
b-lactamases in Enterobacteriaceae. J Antimicrob Chemother 2010; 65:
490–5.
12 Huang XZ, Cash DM, Chahine MA et al. Development and validation of
a multiplex TaqMan real-time PCR for rapid detection of genes encoding
four types of class D carbapenemase in Acinetobacter baumannii.J Med
Microbiol 2012; 61: 1532– 7.
13 Naas T, Ergani A, Carrer A et al. Real-time PCR for detection of NDM-1
carbapenemase genes from spiked stool samples. Antimicrob Agents
Chemother 2011; 55: 4038– 43.
14 Poirel L, Walsh TR, Cuvillier V et al. Multiplex PCR for detection of
acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011; 70:
119–23.
15 Swayne RL, Ludlam HA, Shet VG et al. Real-time TaqMan PCR for rapid
detection of genes encoding five types of non-metallo- (class A and D)
carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents 2011;
38:358.
16 ISO 15189:2012. Medical Laboratories – Requirements for Quality
and Competence. http://www.iso.org/iso/home/store/catalogue_ics/
Accredited PCR for carbapenemases and minor ESBLs
1581
JA
C
at Universidade Federal do Par� on June 14, 2016http://jac.oxfordjournals.org/Downloaded from
catalogue_detail_ics.htm?csnumber=56115 (2 January 2013, date last
accessed).
17 Naas T, Poirel L, Karim A et al. Molecular characterization of In50, a
class 1 integron encoding the gene for the extended-spectrum
b-lactamase VEB-1 in Pseudomonas aeruginosa.FEMS Microbiol Lett
1999; 176: 411– 9.
18 Rabenau HF, Kessler HH, Kortenbusch M et al. Verification and
validation of diagnostic laboratory tests in clinical virology. J Clin Virol
2007; 40:938.
19 Naas T, Cotellon G, Ergani A et al. Real-time PCR for detection
of bla
OXA-48
genes from stools. J Antimicrob Chemother 2012; 68:
101–4.
Bogaerts et al.
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... 1.5% agarose gel stained with GreenSafe DNA Gel Stain 5% (Canvax, Cordoba, Spain), and thereafter excised from the gel, purified using the E.Z.N.A Gel Extraction Kit (Omega Bio Tek, Norcross, GA, USA) and sequenced (Macrogen, Seoul, South Korea). In ESBLpositive isolates in which no bla CTX-M , bla SHV , bla TEM or bla OXA-5/10-like was detected, the PCRs were performed twice, in order to avoid false negatives, and the presence of less-frequent ESBLs including bla BEL , bla GES , bla-PER and bla VEB was also established by PCR following previously described procedures (Bogaerts et al. 2013) ( Table 1). ...
... In the carbapenem non-susceptible isolates, the presence of bla KPC , bla NDM , bla IMI , bla VIM , bla IMP , bla GES , bla OXA-48-like , bla SIM , bla SPM and bla GIM was determined by PCR using the primers and conditions reported in Table 1 (Bogaerts et al. 2013;Ellington et al. 2007;Mlynarcik et al. 2016). ...
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This study aimed to analyze Escherichia coli from marketed meat samples in Peru. Sixty-six E. coli isolates were recovered from 21 meat samples (14 chicken, 7 beef), and antimicrobial resistance levels and the presence of mechanisms of antibiotic resistance, as well as clonal relationships and phylogeny of colistin-resistant isolates, were established. High levels of antimicrobial resistance were detected, with 93.9% of isolates being multi-drug resistant (MDR) and 76.2% of samples possessing colistin-resistant E. coli; of these, 6 samples from 6 chicken samples presenting mcr-1-producer E. coli. Colistin-resistant isolates were classified into 22 clonal groups, while phylogroup A (15 isolates) was the most common. Extended-spectrum β-lactamase- and pAmpC-producing E. coli were found in 18 and 8 samples respectively, with blaCTX-M-55 (28 isolates; 16 samples) and blaCIT (8 isolates; 7 samples) being the most common of each type. Additionally, blaCTX-M-15, blaCTX-M-65, blaSHV-27, blaOXA-5/10-like, blaDHA, blaEBC and narrow-spectrum blaTEM were detected. In addition, 5 blaCTX-M remained unidentified, and no sought ESBL-encoding gene was detected in other 6 ESBL-producer isolates. The tetA, tetE and tetX genes were found in tigecycline-resistant isolates. This study highlights the presence of MDR E. coli in Peruvian food-chain. The high relevance of CTX-M-55, the dissemination through the food-chain of pAmpC, as well as the high frequency of unrelated colistin-resistant isolates is reported.
... The amplicons were analyzed and visualized using a DNA ladder via gel electrophoresis on 1.5% (w/v) agarose gel in 0.5 x TAE buffer at a constant voltage of 100 V for 30 min. (Bogaerts et al., 2013) ...
Article
Aims: To determine carbapenem-resistant genes associated to hospital-acquired Gram-negative bacteria pathogens from a tertiary hospital in Lagos, Nigeria. Study Design: Prospective cross-sectional study. Place and Duration of Study: Lagos State University Teaching Hospital Ikeja, Lagos and Nigerian Institute of Medical Research (NIMR), Lagos, between February 2023 and April 2024. Methodology: We collected 162 Gram-negative bacteria isolates from urine and wound cultures of patients suspected of having hospital-acquired infections. Phenotypic identification of the isolates was by standard microbiological procedures. Carbapenem resistance was determined by Kirby-Baurer disc diffusion method using Meropenem and Imipenem. Confirmation of the strains of carbapenem-resistant bacteria isolates was by gene sequencing. The carbapenem-resistant genes of the bacterial strains were detected by conventional Polymerase Chain Reaction (PCR) using carbapenemase primers. Results: Out of 162 bacterial isolates, 13(8%) bacterial isolates were resistant to Meropenem and Imipenem. New Dehli Metallo-β-lactamase (blaNDM) was detected in 6(46%) of the carbapenem-resistant isolates while Verona integron-encoded Metallo-β-lactamase (blaVIM) was detected in 2(15%). Carbapenemase genes were detected in 54% (7/13) of the carbapenem-resistant isolates. A strain of Enterobacter hormaechei harboured blaVIM and blaNDM genes. Conclusion: The presence of these carbapenem-resistant genes in these pathogens is a public health threat. This study has provided knowledge of carbapenem-resistant genotypes of hospital-acquired bacterial pathogens in the study population. There should be renewed clinical monitoring of these pathogens and further studies on other mechanisms of carbapenem resistance.
... We considered CR when non-susceptibility to at least one carbapenem was documented. As a reference standard for carbapenemase identification, polymerase chain reaction (PCR) (Integrated DNA Technologies, Coralville, IA, USA) [30] was used. ...
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Background: Infections due to carbapenem-resistant Gram-negative bacteria are emerging as an important challenge in health-care settings and a growing concern worldwide. Lateral flow immunoassay NG-Test® CARBA 5 can detect the five most reported carbapenemases (KPC, OXA-48-like, VIM, IMP, and NDM). Direct testing of positive blood cultures could reduce time to detection. This study aims to validate and report on the diagnostic yield of a novel method for carbapenemase detection in positive blood culture vials using NG-Test® CARBA 5. Methods: We implemented an investigator-developed method for the direct testing of positive blood cultures using NG-Test® CARBA 5. We compared results between genotypic, phenotypic, and direct NG-Test® CARBA 5 in blood. Results: A total of 32 isolates were tested (21 Enterobacterales and 11 Pseudomonas aeruginosa). Genotypic testing detected 23 carbapenemases. When comparing the results of NG-Test® CARBA 5 in blood with genotypic testing, agreement was observed in 31/32 (97%) tests. The sensitivity, specificity, positive predictive value, and negative predictive value of the NG-Test® CARBA 5 in blood were 93%, 100%, 100%, and 94%, respectively. Conclusions: Our method using NG-Test® CARBA 5 directly in blood culture samples presented an excellent diagnostic yield when compared to genotypic profiling and permits an accurate detection of carbapenemases.
... All the strains were screened for carbapenemase genes using either the Coris rapid immunochromatographic test Coris BioConcept,Gembloux,Belgium) or the Cepheid Carba-R assay on the GeneXpert platform (Cepheid, Sunnyvale, CA, USA). If negative, the strains were tested with an in-house PCR (ISO15189 accredited) targeting bla VIM , bla IMP , bla NDM , bla KPC , and bla OXA-48 and PCR OXACARBA targeting bla OXA-23 , bla OXA-24 , and bla OXA-58 (Bogaerts et al., 2013). All three techniques have the ability to detect the OXA-48-like, KPC, NDM, VIM, and IMP genes/proteins. ...
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Introduction Antimicrobial susceptibility testing (AST) using broth microdilution (BMD) is usually the reference method to obtain accurate minimum inhibitory concentrations and optimally manage infections with resistant organisms. Several commercial dry BMD are available for AST in clinical laboratories. Materials and methods Two commercial BMD panels for testing of multidrug-resistant Gram-negative bacteria were compared: the Thermo Scientific™ Sensititre DKMGN and the Beckman Coulter NMDRM1, for 17 antimicrobial agents. Results A total of 207 isolates were tested: three ATCC strains and one NCTC strain, six quality control strains from the Belgian National Antimicrobial Committee, and 197 clinical isolates, including carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 breakpoints version 13.1 were used to assign susceptibility categories. Discussion Overall, the categorical agreement (CA) and essential agreement (EA) were both above 90%, but several useful antibiotics for the treatment of multi-resistant organisms showed CA and EA under 90%, that is, meropenem, imipenem, and colistin for Enterobacterales and meropenem and colistin for P. aeruginosa. For Enterobacterales, the NMDRM1 panel showed a significantly higher resistance rate for meropenem, imipenem, amikacin, and colistin. For carbapenems, the minimal inhibitory concentrations (MICs) were underestimated by the DKMGN panel, as already pointed out by a warning on the EUCAST website. To better assess carbapenem susceptibility in carbapenem-resistant organisms, the DKMGN panel now requires the use of a higher inoculum in the insert kit. However, for a given isolate whose susceptibility to carbapenems is not known, there is a risk of underestimating the MIC values. Our results show that colistin testing remains a challenge, highlighting the urgent need for the development of more accurate commercial methods. The use of a single commercial method cannot guarantee good precision in the determination of the MIC value for colistin.
... Subsequently, multiplex PCR was performed to assess DNA from each isolate, followed by confirmation through single PCR. Detection of clinically significant carbapenemase genes (bla NDM , bla KPC , bla IMP , bla VIM , bla OXA-48 , and bla GES ) was conducted using the specific primers outlined in Table 1 [66,67]. The identification of carbapenemase genes utilized GoTaq ® DNA polymerase (Promega, Madison, WI, USA) and a T100 thermal cycler (Bio-Rad, Hercules, CA, USA). ...
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Wastewater treatment plants (WWTPs) are recognized as important niches of antibiotic-resistant bacteria that can be easily spread to the environment. In this study, we collected wastewater samples from the WWTP of A Coruña (NW Spain) from April 2020 to February 2022 to evaluate the presence of Gram-negative bacteria harboring carbapenemase genes. Bacteria isolated from wastewater were classified and their antimicrobial profiles were determined. In total, 252 Gram-negative bacteria carrying various carbapenemase genes were described. Whole-genome sequencing was conducted on 55 selected carbapenemase producing isolates using Oxford Nanopore technology. This study revealed the presence of a significant population of bacteria carrying carbapenemase genes in WWTP, which constitutes a public health problem due to their risk of dissemination to the environment. This emphasizes the usefulness of WWTP monitoring for combating antibiotic resistance. Data revealed the presence of different types of sequences harboring carbapenemase genes, such as blaKPC-2, blaGES-5, blaGES-6, blaIMP-11, blaIMP-28, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-217, and blaVIM-2. Importantly, the presence of the blaKPC-2 gene in wastewater, several months before any clinical case was detected in University Hospital of A Coruña, suggests that wastewater-based epidemiology can be used as an early warning system for the surveillance of antibiotic-resistant bacteria.
... Bacterial isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Biotyper; Bruker Daltonics, Bremen, Germany). When CPO was suspected on rectal swabs or clinical samples based on antimicrobial susceptibility testing, the identification of carbapenemase type was confirmed by an in-house multiplex polymerase chain reaction or by immunochromatographic assay (K-SeT; Coris BioConcept, Gembloux, Belgium) [11]. ...
... The bacterial samples on the microplate were then analyzed using MALDI-TOF MS, and the isolate was classified at the genus and species level using the MALDI Biotyper® software and further confirmed by 16S rDNA sequencing. In the group of carbapenem-resistant strain, the presence of the carbapenemase genes including bla NDM , bla IMP , bla VIM , bla KPC , and bla OXA was determined by using PCR as previously described (Bogaerts et al. 2013;Ellington et al. 2007). ...
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The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) blaNDM-5Escherichia coli and 40 (9.1%) blaKPC-2Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5E. coli, while all blaKPC-2 K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including blaCTX-M, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that blaNDM-5E. coli was highly heterogeneous while the blaKPC-2 K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with blaNDM-5 spread, while IncFII-R and IncFII plasmids mediated blaKPC-2 spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including blaKPC-2. In addition, all of the blaKPC-2 K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried blaKPC-2- and pLVKP-like virulence plasmids from migratory birds. The blaKPC-2 K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. Key points • Multidrug resistance plasmids (blaKPC-2, bla436NDM-5, bla CTX-M, qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.
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Purpose Tolerance and persistence occur “silently” in bacteria categorized as susceptible by antimicrobial susceptibility testing in clinical microbiology laboratories. They are different from resistance phenomena, not well-studied, and often remain unnoticeable. We aimed to investigate and characterize ceftazidime-avibactam (CZA) tolerance/persistence in 80 Klebsiella pneumoniae isolates from bloodstream infections. Methods We used the Tolerance Disk Test (TDtest) to detect CZA tolerance/persistence and investigate the avibactam (AVI) influence on them, and time-kill assays with minimal duration for killing (MDK) determination to characterize/differentiate CZA tolerance from persistence, for selected isolates. Whole genome sequencing was performed for 49/80 selected isolates to investigate genes related to beta-lactam tolerance/persistence and resistance as well as phylogeny studies. Results Tolerance/persistence to CZA was detected in 48/80 (60%) isolates, all extensively drug-resistant (XDR) or multidrug-resistant, carbapenem-resistant K. pneumoniae (CRKp), KPC producers, and previously categorized as susceptible (not resistant) to CZA. No heteroresistance was detected. CZA tolerance/persistence occurred due to ceftazidime tolerance/persistence and was not related to AVI in the CZA combination. 5/11 isolates were characterized as CZA-tolerant and 5/11 as CZA-persistent. The single (1/11) XDR and CRKp non-KPC producer was truly susceptible. All the CZA-tolerant/persistent isolates (ST11, ST258, ST340, ST437, ST16, ST17, and ST307) harbored the carbapenemase-encoding gene blaKPC−2. Mutation in only two genes (rpoS and degQ) related to beta-lactam tolerance/persistence was found in only 7/49 CZA-tolerant/persistent isolates, suggesting the presence of yet unknown beta-lactam tolerance/persistence genes. Conclusion Among the K. pneumoniae bloodstream isolates studied, 60%, previously categorized as susceptible to CZA, were, actually, tolerant/persistent to this antibiotic, all these KPC producers.
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Aeromonas veronii, a threatening fish pathogen, is implicated in food-borne infections in humans, contributing to substantial economic losses in the aquaculture sector. To examine the occurrence, resistance profiles, 16SrRNA sequence analysis, virulence-determinant and resistance genes, and pathogenicity of A. veronii isolated from Nile tilapia (Oreochromis niloticus), almost 150 fish weighing 75 ± 10 g (apparently healthy; n = 50 and moribund fish; n = 100) were collected from commercial farms in Ismailia Governorate, Egypt. Subsequently, the clinical, post-mortem, and bacteriological examinations were carried out. The overall prevalence of A. veronii in the collected O. niloticus was 18.6% (28/150), where the pathogen was only detected in the moribund one. Phylogenetic analysis revealed that the tested isolate exhibited significant genetic identity with other strains from diverse locations and origins in China and Egypt. PCR showed that aerA (95.4%) was the most predominant virulence gene associated with A. veronii isolates retrieved from O. niloticus, followed by hly (80.3%), ast and ser (69.7% for each), and alt (50.1%). Moreover, 40.9% of the obtained isolates were extensively drug-resistant (XDR) to seven classes and inherited blaTEM, tetA, blaCTX-M, and sul1 genes. Besides, 6.1% of the recovered isolates were carbapenem-resistant and XDR to seven classes harboring blaTEM, blaNDM, blaCTX-M, sul1, and tetA genes. The cumulative mortality rate was 86.7% in the fish group injected with a virulent A. veronii strain at a concentration of 3 × 10⁸ CFU/mL. Concisely, this study underscored the existence of XDR A. veronii in O. niloticus specifying a public health hazard. XDR A. veronii isolated from O. niloticus commonly inherited aerA, ast, ser, and alt virulence-determinant genes and blaTEM, sul1, blaCTX-M, and tetA resistance genes.
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A biochemical test (Carba NP test II) was developed to identify carbapenemase production in Enterobacteriaceae and Pseudomonas spp. and to discriminate between the different types of carbapenemases (classes A, B, and D). It is based on the detection of the acidification resulting from imipenem hydrolysis, coupled with tazobactam and EDTA as inhibitors. This is an easy and reliable technique (100% sensitivity and specificity) for detection of not only carbapenemase activity but also carbapenemase types in Enterobacteriaceae and Pseudomonas aeruginosa.
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Enterobacterial strains producing clavulanic-acid-inhibited extended-spectrum β-lactamases (ESBLs) are increasingly reported worldwide. Conventional detection of ESBL production remains time-consuming (24 to 48 h). Therefore, the ESBL NDP (Nordmann/Dortet/Poirel) test was developed for a rapid identification of ESBLs in Enterobacteriaceae. This biochemical test was based on the in vitro detection of a cephalosporin (cefotaxime) hydrolysis that is inhibited by tazobactam addition. The ESBL activity was evidenced by a color change (red to yellow) of a pH indicator (red phenol) due to carboxyl-acid formation resulting from cefotaxime hydrolysis that was reversed by addition of tazobactam (positive test). The ESBL NDP test was applied to cultured strains (215 ESBL producers and 40 ESBL nonproducers). Its sensitivity and specificity were 92.6% and 100%, respectively. Its sensitivity (100%) was excellent for detection of CTX-M producers. A few ESBL producers (n = 16) that remained susceptible to cefotaxime were not detected. The test was also evaluated on spiked blood cultures and showed excellent sensitivity and specificity (100% for both). The test was rapid (less than 1 h) and cost-effective. It can be implemented in any health care facility and is well adapted for infection control purposes in particular.
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OXA-48-type carbapenem-hydrolysing class D β-lactamases are increasingly reported in enterobacterial species. To date, six OXA-48-like variants have been identified, with OXA-48 being the most widespread. They differ by a few amino acid substitutions or deletions (one to five amino acids). The enzymes hydrolyse penicillins at a high level and carbapenems at a low level, sparing broad-spectrum cephalosporins, and are not susceptible to β-lactamase inhibitors. When combining permeability defects, OXA-48-like producers may exhibit a high level of resistance to carbapenems. OXA-163 is an exception, hydrolysing broad-spectrum cephalosporins but carbapenems at a very low level, and being susceptible to β-lactamase inhibitors. The bla(OXA-48)-type genes are always plasmid-borne and have been identified in association with insertion sequences involved in their acquisition and expression. The current spread of the bla(OXA-48) gene is mostly linked to the dissemination of a single IncL/M-type self-transferable plasmid of 62 kb that does not carry any additional resistance gene. OXA-48-type carbapenemases have been identified mainly from North African countries, the Middle East, Turkey and India, those areas constituting the most important reservoirs; however, occurrence of OXA-48 producers in European countries is now well documented, with some reported hospital outbreaks. Since many OXA-48-like producers do not exhibit resistance to broad-spectrum cephalosporins, or only decreased susceptibility to carbapenems, their recognition and detection can be challenging. Adequate screening and detection methods are therefore required to prevent and control their dissemination.
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A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram. Preliminary PCR analysis revealed the presence of blaVEB-1, an integron-located gene encoding an extended-spectrum β-lactamase previously identified in Escherichia coli MG-1. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1 containing integron along with some flanking sequence from P. aeruginosa JES was amplified and subsequently sequenced. In50 contains within its variable region, in addition to qacEΔ1 and sul1 genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB. In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000. P. aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries. The finding of blaVEB-1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum β-lactamase gene.
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Objectives: Outbreaks of OXA-48-like carbapenemase producers are increasingly reported in many European countries and are often the result of difficulties in detection, especially for isolates with MICs of carbapenems that remain in the susceptibility range. Methods: An in-house real-time quantitative PCR (qPCR) assay using TaqMan chemistry to detect bla(OXA-48-like) genes was compared with bacterial culturing on ChromID ESBL and SUPERCARBA media of spiked stool samples with several species producing OXA-48 variants. Results: qPCR amplification using plasmid DNA was linear over 10 log dilutions (r(2) = 0.998 and slope = -3.14), with an amplification efficiency of 1.10, and the detection limit of the assay was reproducibly estimated at 10 plasmid molecules/PCR. No cross-reaction was detected with DNA extracted from several multidrug-resistant bacteria harbouring other β-lactam resistance genes. The bla(OXA-48) qPCR assay was capable of detecting 10-50 cfu of OXA-48 producers/100 mg of faeces. ChromID ESBL was capable of detecting OXA-48 producers (1 × 10(1) to 3 × 10(2) cfu/100 mg of faeces), as long as the isolates exhibited a high level of resistance to cephalosporins due to an associated extended-spectrum β-lactamase. The SUPERCARBA screening medium was capable of detecting all the OXA-48-like producers (1-3 × 10(1) cfu/100 mg of faeces), except those producing OXA-163, a variant lacking carbapenem-hydrolysing activity. Conclusions: The qPCR is likely to shorten the time for bla(OXA-48) detection from 48 to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.
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A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D β-lactamases (bla(OXA-23)-like, bla(OXA-24/40)-like, bla(OXA-51)-like and bla(OXA-58)-like genes) was developed and evaluated for early detection of imipenem (IMP) resistance in clinically significant Acinetobacter baumannii isolates. Well-characterized strains of A. baumannii were used as positive controls and non-Acinetobacter strains were used to assess specificity. Analytical sensitivity was quantified by comparison with the number of bacterial c.f.u. Forty of 46 (87 %) clinically significant and IMP-resistant A. baumannii isolates were positive for the bla(OXA-23)-like gene, and one isolate (2 %) was positive for the bla(OXA-58)-like gene. The bla(OXA-24/40)-like gene was not detected in any of the 46 IMP-resistant strains and the bla(OXA-51)-like gene was identified in both IMP-resistant and non-resistant A. baumannii. All 11 non-Acinetobacter bacteria produced a negative result for each of the four bla(OXA) genes. This assay was able to detect as few as 10 c.f.u. per assay. This real-time PCR method demonstrated rapid detection of OXA-like carbapenem resistance in A. baumannii in comparison with phenotypic susceptibility testing methodology. This method could be adapted to a multiplexed single reaction for rapid detection of genes associated with carbapenem resistance in A. baumannii and potentially other clinically significant multidrug-resistant Gram-negative bacteria.
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Beta-lactamase evolution presents to the infectious disease community a major challenge in the treatment of infections caused by multidrug-resistant Gram-negative bacteria. Because over 1,000 of these naturally- occurring beta-lactamases exist, attempts to correlate structure and function have become daunting. Although new enzymes in the extended-spectrum beta-lactamase (ESBL) families are frequently identified, the older CTX-M-14 and CTX-M-15 enzymes have become the most prevalent ESBLs in global surveillance. Carbapenemases with either serine-based or zinc-facilitated hydrolysis mechanisms are posing some of the most critical problems. Most geographical regions now report KPC serine carbapenemases and the metallo-beta-lactamases VIM, IMP, and NDM-1, even though NDM-1 was only recently identified. The rapid emergence of these newer enzymes, with multiple beta-lactamases appearing in a single organism, makes the design of new beta-lactamase inactivators or b-lactamase-stable beta-lactams all the more difficult. Combination therapy will likely be required to counteract the continuing evolution of these insidious enzymes in multidrug-resistant pathogens.
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Antibiotic resistance in Acinetobacter spp., particularly Acinetobacter baumannii, is increasing rapidly. A. baumannii possesses two intrinsic β-lactamase genes, in addition to weak permeability and efflux systems, that together confer a natural reduced susceptibility to antibiotics. In addition, numerous acquired mechanisms of resistance have been identified in A. baumannii. The very high genetic plasticity of A. baumannii allows an accumulation of resistance determinants that give rise to multidrug resistance at an alarming rate. The role of novel genetic elements, such as resistance islands, in concentrating antibiotic resistance genes in A. baumannii requires detailed investigation in the near future.