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Contents lists available at SciVerse ScienceDirect
International Journal of Antimicrobial Agents
journal homepage: http://www.elsevier.com/locate/ijantimicag
Limited diversity in the gene pool allows prediction of
third-generation cephalosporin and aminoglycoside resistance in
Escherichia coli and Klebsiella pneumoniae
Andrew N. Ginn a,b,c , Zhiyong Zong a,d , Agnieszka M. Wiklendt a , Lee C. Thomas a ,
John Merlino e , Thomas Gottlieb e , Sebastiaan van Hal f,1 , Jock Harkness g , Colin Macleod h ,
Sydney M. Bell i , Marcel J. Leroi j , Sally R. Partridge a,b,c , Jonathan R. Iredell a,b,c,∗
a
Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, Sydney, New South Wales, Australia
Centre for Research Excellence in Critical Infection and Sydney Institute for Emerging Infections and Biosecurity, University of Sydney, Sydney, New South
Wales, Australia
c
Westmead Millennium Institute, Westmead, New South Wales, Australia
d
Department of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China
e
Department of Microbiology and Infectious Diseases, Concord Hospital, Sydney, New South Wales, Australia
f
Department of Microbiology and Infectious Diseases, Sydney South West Pathology Service–Liverpool, South Western Sydney Local Health Network,
Sydney, New South Wales, Australia
g
St Vincent’s Hospital, Sydney, Division of Microbiology, SydPath, Darlinghurst, New South Wales, Australia
h
Department of Microbiology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia
i
Department of Microbiology, The Prince of Wales Hospital, Sydney, New South Wales, Australia
j
Department of Pathology, Nepean Hospital, Sydney West Area Health Service, Sydney, New South Wales, Australia
b
a r t i c l e
i n f o
Article history:
Received 6 December 2012
Accepted 12 March 2013
Keywords:
Antibiotic resistance
Diagnostic targets
Enterobacteriaceae
a b s t r a c t
Early appropriate antibiotic treatment reduces mortality in severe sepsis, but current methods to identify
antibiotic resistance still generally rely on bacterial culture. Modern diagnostics promise rapid gene
detection, but the apparent diversity of relevant resistance genes in Enterobacteriaceae is a problem. Local
surveys and analysis of publicly available data sets suggested that the resistance gene pool is dominated by
a relatively small subset of genes, with a very high positive predictive value for phenotype. In this study,
152 Escherichia coli and 115 Klebsiella pneumoniae consecutive isolates with a cefotaxime, ceftriaxone
and/or ceftazidime minimum inhibitory concentration (MIC) of ≥2 ␮g/mL were collected from seven
major hospitals in Sydney (Australia) in 2008–2009. Nearly all of those with a MIC in excess of European
Committee on Antimicrobial Susceptibility Testing (EUCAST) resistance breakpoints contained one or
more representatives of only seven gene types capable of explaining this phenotype, and this included
96% of those with a MIC ≥ 2 ␮g/mL to any one of these drugs. Similarly, 97% of associated gentamicinnon-susceptibility (MIC ≥ 8 ␮g/mL) could be explained by three gene types. In a country like Australia,
with a background prevalence of resistance to third-generation cephalosporins of 5–10%, this equates
to a negative predictive value of >99.5% for non-susceptibility and is therefore suitable for diagnostic
application. This is an important proof-of-principle that should be tested in other geographic locations.
© 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
Delays in effective antibiotic treatment increase mortality in
septic shock [1], with culture-based methods remaining noncontributive for many hours beyond the period of greatest impact.
An assay to exclude antibiotic resistance by quickly detecting
relevant genes would at the very least obviate the need for culture
∗ Corresponding author. Tel.: +61 2 9845 6012; fax: +61 2 9891 5317.
E-mail address: [email protected] (J.R. Iredell).
1
Present address: Royal Prince Alfred Hospital, Missenden Road, Camperdown,
Sydney, New South Wales, Australia.
and provide a useful infection control and/or surveillance tool. If
such an assay could be deployed effectively to test blood at the
point of presentation, it would also prevent the use of unnecessarily broad-spectrum, toxic or inferior antibiotics in life-threatening
sepsis. Modern diagnostic platforms for the detection of relevant
genetic markers are already widely used for the detection of,
for example, meticillin-resistant Staphylococcus aureus [2], but a
diverse pool of transmissible resistance genes in the medically
important Enterobacteriaceae has the capacity to confound such
an approach.
Resistance to the important third-generation cephalosporin
antibiotics in major bacteria such as Escherichia coli and
0924-8579/$ – see front matter © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
http://dx.doi.org/10.1016/j.ijantimicag.2013.03.003
Please cite this article in press as: Ginn AN, et al. Limited diversity in the gene pool allows prediction of third-generation
cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents (2013),
http://dx.doi.org/10.1016/j.ijantimicag.2013.03.003
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2
Klebsiella pneumoniae is most commonly due to ‘classical’
extended-spectrum ␤-lactamase (ESBL) enzymes but also to
plasmid-borne AmpC ␤-lactamases and/or carbapenemases. At
least 12 types (>350 variants) of ESBL genes and 5 types (>100
variants) of plasmid-borne ampC genes can confer resistance
to third-generation cephalosporins in Gram-negative bacteria, as can ca. 30 types (ca. 100 variants) of ‘carbapenemases’
(http://www.lahey.org/Studies/). The aminoglycoside antibiotics
that are often used in combination with ␤-lactams for the treatment of serious sepsis due to Gram-negative bacteria may be
defeated by seven known types of acquired 16S rRNA methylases,
which confer high-level resistance to all clinically relevant aminoglycosides, as well as ca. 50 known aminoglycoside-modifying
enzymes that confer resistance to different subsets and are
encoded by mobile genes [3,4].
It is presently impractical to rapidly screen for all of these genes,
but most published surveys, including our own, suggest that most
resistance may be attributable to a small subset of these genes.
In Sydney, Australia, CTX-M-type ␤-lactamases dominated in isolates resistant to third-generation cephalosporins both in E. coli and
K. pneumoniae [5,6]. Other genes may cause important resistance,
including local plasmid-borne carbapenemases [7].
The aim of this study was to test whether it is possible to identify a small subset of plasmid-borne resistance genes that act as
reliable predictive markers for resistance (and susceptibility) to
third-generation cephalosporins, as well as associated resistance to
aminoglycosides, in E. coli and K. pneumoniae isolates from Sydney.
To enable simultaneous screening of multiple isolates for multiple genes, a multiplex PCR/reverse line blot (mPCR/RLB) assay was
used, which is a robust and relatively cheap system that requires
minimal infrastructure and has been proven in a variety of clinical
microbiological applications [8]. A future aim would be to use the
identified predictive targets to develop rapid tests designed for use
with clinical specimens.
There are minor variations in the way antibiotic resistance is defined, so for the purposes of this study we treated
ceftriaxone (CRO) and cefotaxime (CTX) minimum inhibitory
concentrations (MICs) as equivalent, and focused more on susceptibility than resistance, using breakpoints from the European
Committee on Antimicrobial Susceptibility Testing (EUCAST)
(http://www.eucast.org) and Clinical and Laboratory Standards
Institute (CLSI) (M100-S22, 2012), as it is the ability to predict
antibiotic susceptibility that is most important to the clinician.
2. Materials and methods
2.1. Bacterial strains
In 2008–2009, microbiology laboratories at seven major hospitals in Sydney collected unique clinical isolates of E. coli and K.
pneumoniae (one per patient) for which the MIC, as determined
by the standardised nationally accredited method used in each
laboratory, of CTX, CRO and/or ceftazidime (CAZ) was ≥2 ␮g/mL
(CLSI and EUCAST susceptibility breakpoints are 1 ␮g/mL). A
total of 267 isolates were collected as follows [numbers of E. coli
(N = 152) and K. pneumoniae (N = 115), respectively]: Westmead
Hospital: n = 35, n = 32; Concord Hospital: n = 37, n = 23; Royal
Prince Alfred Hospital, n = 22, n = 19; St Vincent’s Hospital, n = 20,
n = 12; Liverpool Hospital, n = 15, n = 15; Nepean Hospital, n = 13,
n = 6; and Prince of Wales Hospital, n = 10, n = 8. MICs were re-tested
using a Phoenix automated microbiology system (NMIC/ID-101
AST; Becton Dickinson, Sparks, MD) and/or by Etest (bioMérieux
Diagnostics, Marcy-l’Étoile, France) for all isolates where discrepancies emerged between the presence of a gene and a resistance
phenotype. Isolates with no gene detected to explain an elevated
CTX/CRO/CAZ MIC were further examined by disk diffusion with
clavulanic acid for an ESBL phenotype [9] and/or with boronic acid
for an AmpC phenotype [10].
Cells were harvested from overnight growth on blood agar
(Oxoid Ltd., Basingstoke, UK) incubated at 37 ◦ C in 5% CO2 , resuspended in 1 mL of phosphate-buffered saline, boiled for 10 min
and centrifuged for 2 min at 13 000 × g. Lysates prepared in this way
were stored at −80 ◦ C for use as templates in the mPCR/RLB assay
and other PCRs.
2.2. Design of the multiplex PCR/reverse line blot assay
Forty-two primers [18–29 nt; melting temperature
(Tm ) = 58.5–65.5 ◦ C], predicted to produce amplicons of
123–377 bp, and 22 probes (19–30 nt; Tm = 63–72.5 ◦ C) were
designed (Table 1). Antisense primers (compared to corresponding
probes) were biotinylated at the 5 -end and probes were 5 -labelled
with an amine group.
Primers and probes to detect common bla genes were designed
from alignment of sequences from GenBank accession nos. listed
at http://www.lahey.org/Studies/ and were re-checked against all
sequences in this database in February 2013. A single pair of primers
and a probe designed with the intention of detecting most of
the >130 known blaCTX-M variants have single mismatches from
blaCTX-M-33 , -67 , -74 , -75 and -114 . The blaTEM primers and probe were
designed outside the regions containing mutations resulting in different ‘frameworks’ [21] with the aim of detecting all known blaTEM
genes. Only blaTEM-193 , reported since this assay was developed, has
a single mismatch from the reverse primer. Primers expected to
amplify most of the known >160 blaSHV variants were used, but to
avoid detecting chromosomal blaSHV genes present in the majority
of K. pneumoniae, a probe targeting only those with the G700A and
G703A mutations resulting in Gly238Ser and Glu240Lys substitutions found in SHV-5 and SHV-12 [22], both previously detected
locally, was used. Many, but not necessarily all, ESBL-type SHVs
would be expected to be detected by this probe.
blaCMY-2 -like and blaDHA genes, encoding the most commonly
encountered AmpC enzymes, were included as targets, and the
respective primer/probe sets are expected to detect all known variants of each gene at the time of writing. The blaIMP primers/probe
are expected to detect all variants for which sequences are
available, except blaIMP-27 (mismatches close to the 3 -end of the
forward primer and probe). The blaVIM primers and probe are
expected to detect all variants for which sequences are available,
except possibly blaVIM-27 and blaVIM-37 (single mismatch in the
forward primer). The blaKPC primers and probes are expected to
detect all known variants.
Other genes that may more rarely account for an EBSL
phenotype were also included in the assay. The blaOXA-10 -like
primer/probes are expected to detect all known blaOXA-10 (-11 , -14 ,
-16 , -17 , -74 , -142 , -240 , -251 , -256 ) and blaOXA-13 (-7 , -19 , -28 , -35 , -56 , -101 ,
-145 , -147 , -183 ) variants, which include some ESBL types. As blaVEB
(GenBank accession no. EU259884) and blaOXA-23 -like (unpublished
data) genes have been observed locally in Enterobacteriaceae,
primers and probes to detect all known variants (blaVEB-1 –blaVEB-13 ;
blaOXA-23 , -27 , -49 , -73 , -133 , -146 , -165–171 , -225 , -239 ) were designed.
Primers and a probe to detect blaOXA-30 and all known variants
(blaOXA-4 , -31 , -33 , -47 and -224 ) were also included, although this
gene does not give a classical ESBL phenotype.
Primers and probes were designed to amplify the aacA4 [aac(6 )Ib] gene, one of the most common gene cassette-borne genes in
GenBank [3], but also encompass other genes in the aacA4 subgroup
of aac(6 )-Ib/aac(6 )-II aminoglycoside acetyltransferases (aacA3,
aacA5, aacA8, aacA27, aacA31, aacA32, aacA35 and aacA44 genes,
as defined in and with accession nos. from ref [3], and potentially
aacA40, with a single mismatch in the probe). The aac(6 )-Ib-cr
variant (simplified here to aacA4cr) confers reduced resistance to
Please cite this article in press as: Ginn AN, et al. Limited diversity in the gene pool allows prediction of third-generation
cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents (2013),
http://dx.doi.org/10.1016/j.ijantimicag.2013.03.003
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3
Table 1
Oligonucleotides used for multiplex PCR/reverse line blot (mPCR/RLB) assay.
Target
Primer/probea
Sequence (5 –3 )
blaSHV-5/12- type
SHV-F1n
SHV-URb
SHV/ESBL-Z
GCTGGGAAACGGAACTGA
CACRATSCGCTCTGSTTTGTTATT
ACCGGAGCTAGCAAGCGG
blaCTX-M
CTX-U1b
CTX-URn
CTX-UZ
ATGTGCAGYACCAGTAARGTKATGGC
CCVCCVASVTGRGMAATCARYTTRTTCAT
ACGTGYTTYTCVGCAATSGGRTTRTAGTTA
blaTEM
TEM-Fb
TEM-URn
TEM-UZ
ACAGCGGTAAGATYCTTGAG
CTCCGATCGTTGTCAGAAGT
GATGCTTTTCTGTGACTGGTGAG
VEB-UFn
VEB-URb
VEB-UZ
CCCTCAAGACCTTTTGCCTA
TTCCAATCCTTGTGCATTTG
TTCAATCAAAGCAAACGAAGAA
blaIMP
IMP-UFn
IMP-URb
IMP-UZ
ACAYGGYTTGGTGGTTCTTGT
AGTTCATTKGTTAATTCAGANGCATA
GGAATAGAGTGGCTTAAYTCTCRATC
blaVIM
VIM-UFb
VIM-UZ
VIM-URn
TTTGGTCGCATATCGCAAC
TGCTTYTCAATCTCCGCGA
CGTCATGRAAGTGCGTRGA
KPC-Fn
KPC-Rb
KPC-Z
CAGCTCATTCAAGGGCTTTC
GGCGGCGTTATCACTGTATT
ATCTGACAACAGGCATGACG
blaCMY-2 -like
CMY2-Fb
CMY2-Rn
CMY2-Z
CGATATCGTTAAYCGCACCA
CGGATCGCTGAGCTTRATTT
GAATAGCCTGCTCCTGCATC
blaDHA -like
DHA-Fb
DHA-Rn
DHA-Z
ACACTGATTTCCGCTCTGCT
ATTTTCAGTGACCGGCTGTT
GGAATATCCTGCTGTGCCAT
OXA10-Fn
OXA10-Rb
OXA10-Z
GGGAACTGAGTCAAATCCTG
GATTTTCTTAGCGGCAACTTAb
TTTACTTTTTCGCCTTTAACATGGAT
blaOXA-23 -like
OXA23-Fn
OXA23-Rb
OXA23-Z
CCCAATTAGCACATACACAGC
ATTTCTGACCGCATTTCCAT
AGCAGCCAGATGGAAAAATTGT
blaOXA-30 -like
OXA1-Fn
OXA1-Rb
OXA1-Z
CAAATTCAATTCCTGCGTAA
AATAAACCCTTCAAACCATCCc
AAACTGTATGGGAAAACTGGTGC
aac(3)-II.Fb
aac(3)-II.Rn
aac3II-Z
CGTATGAGATGCCGATGC
AAGATAGGTGACGCCGAAC
AAGCAATCGAGAATGCCGTT
aac6II-Fb
aac6II-Rn
aac6II-Z
aac6II/Ib-UZ
CACATHGTYGARTGGTGGG
AABCCBGCCTTCTCRTAGCA
GTYTCDTCYTCCCACCAKCCRYC
GTYTCDTCYTCCCACCDKCCRYC
aadB
aadB-Fb
aadB-Rn
aadB-Z
ACACAACGCAGGTCACATTd
CTAAGAATCCATAGTCCAACTCCe
ATTTCGCTCATCTGCCGCAGCTA
aacC1
aacC1-Fb
aacC1-Rn
aacC1-Z
ATCATTCGCACATGTAGGC
CGGAGACTGCGAGATCATA
CGACCGAAAAGATCAAGAGC
armA
armA-Fn
armA-Rb
armA-Z
GCATCAAATATGGGGGTCTTA
TTGAAGCCACAACCAAAATCT
CGAATGAAAGAGTCGCAACA
rmtC
RMTC-F2n
RMTC-Rb
RMTC-Z
CCCAGAGGGTATTGTTGTCA
ATCCCAACATCTCTCCCACT
ACTCTACTCGATCGGCAGGA
qnrA
QnrA-Fb
QnrA-R2n
QnrA-Z
TTCAGCAAGAGGATTTCTCACf
ATCGCAYTCCCTGAACTCTAT
AAACTGCAATCCTCGAAACTGGC
QnrB-Fb
QnrB-Rn
QnrB-Z
GATCGTGAAAGCCAGAAAGG
CCACAGCTCACACTTTTCCA
TTTRAAAATGGCATCTTTCAGCAT
blaVEB
blaKPC
blaOXA-10 -like
aac(3)-II
aac(6 )
qnrB
Conc. (mM)
nt position
Reference
AF148850
AF148850
X98105
484–501
770–747
764–781
This work
This work
This work
0.6
AY458016
AY458016
AY458016
21018–20993
20789–20817
20882–20911
[11]
This work
This work
2
X54607
X54607
X54607
363–382
638–619
545–523
This work
This work
This work
0.6
AF010416
AF010416
AF010416
451–470
687–668
646–667
This work
This work
This work
1.4
S71932
S71932
S71932
600–620
815–790
754–779
This work
This work
This work
5.4
Y18050
Y18050
Y18050
3382–3400
3529–3511
3576–3558
This work
This work
This work
0.6
AY034847
AY034847
AY034847
209–228
404–385
337–356
This work
This work
This work
0.6
X91840
X91840
X91840
2010–2029
2247–2228
2059–2040
This work
This work
This work
0.6
Y16410
Y16410
Y16410
1008–1027
1214–1195
1120–1101
This work
This work
This work
0.6
U37105
U37105
U37105
1939–1958
2061–2041
2001–2026
This work
[12]
This work
3
AJ132105
AJ132105
AJ132105
1516–1536
1732–1713
1675–1696
This work
[13]
This work
2
AF255921
AF255921
AF255921
1882–1901
2064–2044
1990–2012
This work
[14]
This work
2
X13543
X13543
X13543
739–756
971–953
826–807
[15]
[15]
This work
1.4
5
M29695
M29695
M29695
M29695
791–809
1167–1148
981–959
981–959
This work
This work
This work
This work
2
L06418
L06418
L06418
1300–1318
1527–1505
1355–1333
[16]
[17]
This work
1
U90945
U90945
U90945
1650–1668
1917–1899
1720–1701
This work
This work
This work
1
AF550415
AF550415
AF550415
72528–72548
72737–72717
72641–72660
This work
This work
This work
0.6
AB194779
AB194779
AB194779
7436–7455
7636–7617
7521–7540
This work
[18]
This work
0.6
AY070235
AY070235
AY070235
325–345
581–561
445–423
[19]
This work
This work
3
DQ351241
DQ351241
DQ351241
175–194
447–428
237–214
[20]
This work
This work
10
GenBank
Please cite this article in press as: Ginn AN, et al. Limited diversity in the gene pool allows prediction of third-generation
cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents (2013),
http://dx.doi.org/10.1016/j.ijantimicag.2013.03.003
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4
Table 1 (Continued)
Target
Primer/probea
Sequence (5 –3 )
qnrS
QnrS-UFn
QnrS-URb
QnrS-UZ
TGAACAGGGTGATATCGAAGG
AGTTTGYTCGGGAAAAGTTGG
GATGCAAGTTTCCAACAATGC
Conc. (mM)
GenBank
nt position
Reference
2
AB187515
AB187515
AB187515
9883–9903
10046–10026
9932–9952
This work
This work
This work
F, forward primer (with respect to the direction of transcription of the gene); R, reverse primer; b, primer labelled with biotin at the 5 -end; n, unlabelled primer; Z,
probe. Primers and probes were designed as part of this study except where indicated.
b
OXA10-Rb modified (by adding G at the 5 -end and deleting C at the 3 -end) from primer OPR2 in [12].
c
Modified (by adding AAT at the 5 end) from primer OXA-R in [14].
d
Modified (by deleting G at the 5 -end) from the forward primer used to detect aadB genes in [16].
e
Named NVC71 in [17].
f
Modified (by adding T at the 5 -end and C at the 3 -end) from the forward qnr primer in [19].
a
aminoglycosides but low-level resistance to fluoroquinolones [3].
All known examples of this variant have a G-to-T mutation at position 514 of the aacA4 gene cassette plus either a T-to-A or T-to-C
synonymous mutation at position 283. The antisense aac6II/Ib-UZ
(U = ‘universal’) probe has A, T or G to bind to T, A or C at position
283, whilst the antisense aac6II-Z probe has an A to bind to T only.
Hybridisation only to aac6II/Ib-UZ indicates that an aacA4cr variant is present, whilst hybridisation to both probes indicates than
a non-cr aacA4 variant or another aacA4 subgroup gene is present.
Primers and probes designed to detect all five variants (a–e) of the
common aac(3)-II gene (accession nos. in Fig. 9 of ref. [21]), the gene
cassette-borne aadB and aacC1 genes (accession nos. in ref. [3]), the
locally detected rmtC gene (GenBank accession no. EU144360) and
the widespread armA [4] gene were also included.
Although we did not attempt to define targets to predict quinolone resistance, primers and probes designed to
detect plasmid-mediated quinolone resistance determinants
(http://www.lahey.org/qnrStudies/; accessed February 2013) were
included. These are often associated with ESBL genes but usually
only confer low-level resistance. The respective primers and probes
perfectly match all known qnrA and qnrS variants, whilst the qnrB
primers and/or probe have one to four mismatches to several variants, but were demonstrated to detect at least qnrB1, qnrB2 and
qnrB4 (Table 2).
2.3. Multiplex PCR/reverse line blot assay
The mPCR/RLB assay was performed essentially as described
previously [8]. Multiplex PCR was carried out in 25 ␮L volumes containing 1× reaction buffer, 1 U of HotStarTaq DNA Polymerase (QIAGEN, Hilden, Germany), 3 mM MgCl2 , 200 ␮M of each dNTP, 250 nM
of each primer and 2 ␮L of lysate as template. PCR conditions were
95 ◦ C for 15 min, followed by 35 cycles of 94 ◦ C for 30 s, 55 ◦ C for
30 s and 72 ◦ C for 1 min, and a final elongation at 72 ◦ C for 10 min.
Probes diluted in 180 ␮L of 0.5 M NaHCO3 (pH 8.4) were incubated with a Biodyne® C nylon membrane (Pall Corporation,
Port Washington, NY; 0.45 ␮m pore size) activated in 20 mL of
16% (w/v) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a
45-lane Miniblotter unit (Immunetics Inc., Boston, MA) at room
temperature for 5 min. After removal of residual probe solutions, the membrane was incubated in 250 mL of 0.1 M NaOH
at room temperature for 9 min, washed in 250 mL of 2× SSPE
(saline–sodium phosphate–ethylene diamine tetra-acetic acid
buffer) (Astral Scientific, Gymea, NSW, Australia) and then incubated in 250 mL of 2× SSPE/0.1% SDS (sodium dodecyl sulphate) at
60 ◦ C for 5 min. Diluted multiplex PCR products (20 ␮L in 150 ␮L
of 2× SSPE/0.1% SDS) were denatured and 145 ␮L was loaded onto
the membrane. After hybridisation at 60 ◦ C for 1 h, the membrane
was washed twice in 250 mL of pre-warmed 2× SSPE/0.5% SDS
at 60 ◦ C for 10 min before incubation at 42 ◦ C for 60 min with
streptavidin–peroxidase conjugate (1.5 U) in 15 mL of pre-warmed
2× SSPE/0.5% SDS. After washing (twice in 250 mL of pre-warmed
2× SSPE/0.5% SDS, 42 ◦ C, 10 min, twice in 250 mL of 2× SSPE, room
temperature, 5 min), the membrane was incubated in 15 mL of ECL
Detection Reagent (GE Healthcare, Little Chalfont, UK) for 2 min and
exposed to Hyperfilm® ECL (GE Healthcare) for 1–10 min.
2.4. Validation of the multiplex PCR/reverse line blot assay with
control isolates
Control strains known to carry one or more genes targeted by
the mPCR/RLB assay were obtained from local surveillance studies,
including several where complete plasmids had been sequenced, or
were kindly provided by J. Bell (SA Pathology, Adelaide, Australia)
(Table 2). The amount of each probe used (0.6–10 mM) was optimised to produce relatively consistent spot intensities across all
targets. Where necessary, mPCR/RLB assay results were confirmed
by individual PCR and sequencing, using published primers where
available. Some hybridisation to the blaTEM probe was seen in samples that did not have a blaTEM gene by other methods, but this
is consistent with reported contamination of commercial preparations of Taq DNA Polymerase with vector DNA carrying the blaTEM-1a
gene [25], and true positives were easily distinguishable.
2.5. Additional PCR
Published multiplex PCR assays were used to confirm the presence of blaSHV-5/12 -like variants and to distinguish blaCTX-M-1 group
and blaCTX-M-9 group genes [5] as well as to amplify other blaCTX
gene types for sequencing [26]. A multiplex PCR assay [27] was
used to confirm the presence of blaCMY-2 -like and blaDHA -like genes
and to detect the four other known ampC gene types. Published
primers were used to amplify complete blaTEM genes [28] and E. coli
chromosomal ampC promoter regions [29] for sequencing.
2.6. Estimation of negative predictive values (NPVs) at difference
prevalence rates
The NPVs of the mPCR/RLB assay for non-susceptibility to thirdgeneration cephalosporins (here, CTX/CRO/CAZ MIC ≥ 2 ␮g/mL) or
to gentamicin (MIC ≥ 8 ␮g/mL) were calculated. Defining the number of isolates with the phenotype of interest as P and assuming a
prevalence rate of X%, the estimated total number of isolates (=T)
would be (100P/X). Defining the portion of P for which an explanatory gene was not detected (false negatives) as U, the NPV of the
assay is the number of isolates without the phenotype (true negatives) divided by the total number of isolates giving no results in
the assay (true negatives + false negatives) = (T − P)/(T − P + U).
3. Results
3.1. Detection of genes that could explain non-susceptibility to
cefotaxime or ceftazidime by multiplex PCR/reverse line blot assay
On initial screening, the mPCR/RLB assay revealed at least one
known ‘classical’ ESBL gene (blaCTX-M variant and/or blaSHV-5/12 )
Please cite this article in press as: Ginn AN, et al. Limited diversity in the gene pool allows prediction of third-generation
cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents (2013),
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5
Table 2
Control isolates used to validate the multiplex PCR/reverse line blot (mPCR/RLB) assay.
Isolate
Species
Resistance genesa
Reference/sourceb
2.64
LT12
JIE014
JIE058
JIE101
JIE117
JIE137
JIE142
JIE146
JIE161
JIE203
JIE216
JIE242
JIE248
JIE273
JIE275
JIE419
El1573
N13283
N12282
N5249
N3764
N12636
WM99c
KPN2303
Enterobacter gergoviae
Klebsiella pneumoniae
K. pneumoniae
Escherichia coli
E. coli
E. coli
K. pneumoniae
K. pneumoniae
K. pneumoniae
E. coli
K. pneumoniae
E. coli
E. coli
Enterobacter cloacae
Proteus mirabilis
E. coli
E. coli
E. cloacae
K. pneumoniae
E. cloacae
K. pneumoniae
Pseudomonas aeruginosa
P. aeruginosa
Acinetobacter baumannii
K. pneumoniae
blaSHV-140 , aacA27
blaTEM-1b , blaOXA-10 , aadB
blaCTX-M-14 , blaTEM-1b , qnrS1 [aac(3)-II]
blaCTX-M-27 , blaTEM-1b [aac(3)-II]
blaCTX-M-15 , blaOXA-30 , blaTEM-1b , aac(3)-IIe, aacA4cr
blaCMY-2 , blaTEM-1b [aac(3)-II]
blaCTX-M-62 , qnrB2
blaDHA-1 , blaSHV-11 , blaOXA-10
blaCTX-M-15 , blaSHV-11 , blaTEM-1b , blaOXA-30 , aac(3)-IIe, aacA4cr, qnrB1
blaCTX-M-3 , blaTEM-1b [aac(3)-II]
blaDHA-1 , blaSHV-109 , blaTEM-1b , aac(3)-IId, qnrB4
blaCTX-M-24 , blaTEM-1b [aac(3)-II]
blaCTX-M-15 , blaTEM-1b , aac(3)-IId
blaSHV-12 , blaTEM-1b , aacA27, qnrA1
blaVEB-6 , aacA4, aadB, rmtC
blaTEM-1b , armA
blaCMY-42 (aacA4cr, blaOXA-30 -like)
blaIMP-4 , blaTEM-1b , aac(3)-IId, aacA4, qnrB2
blaIMP-1 [blaOXA-30 -like, aac(6 )]
blaIMP-8 , blaSHV-12 [aac(6 ), blaTEM , qnrB]
blaIMP-11
blaVIM-2 , aacA4
blaVIM-3 [aac(6 ), aadB]
blaOXA-23 , aacC1 (blaTEM )
blaKPC-2 (blaSHV-5/12 )
WMH
HQ247816
[6]
[6]
EU418922
WMH
EF219134
WMH
[6]
[6]
WMH
[6]
WMH
WMH
EU144360, EU259884
WMH
WMH
JX101693
[23]
J. Bell
J. Bell
J. Bell
J. Bell
EF015499, EF015500
[24]
a
Genes/variants confirmed by sequencing unless shown in parentheses. blaCTX-M-3 , -15 and -62 belong to the blaCTX-M-1 group. blaCTX-M-14 , -24 and -27 belong to the blaCTX-M-9
group. blaCMY-42 is a blaCMY-2 -like gene. blaSHV-11 , -109 and -140 do not have the mutations associated with blaSHV-5/12 , do not encode extended-spectrum ␤-lactamase (ESBL)
variants of SHV and were not detected by the blaSHV-5/12 -specific probe used in the mPCR/RLB assay. aac(6 ) indicates a gene in the aacA4 subgroup of aminoglycoside
acetyltransferases (see Section 2.2).
b
GenBank accession nos. or references are given when possible. WMH, Westmead Hospital, unpublished; J. Bell, obtained from Jan Bell (SA Pathology, Adelaide, Australia).
Table 3
Genes identified in isolates with cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentrations of ≥2 ␮g/mL.
Gene type
Gene
Total (N = 267)
Escherichia coli (n = 152)
Klebsiella pneumoniae (n = 115)
ESBL (n = 208)
blaCTX-M-1 group only
blaCTX-M-1 + blaCTX-M-9 groups
blaCTX-M-1 group + blaSHV-5/12 -type
blaCTX-M-1 group + blaCMY-2 -like
blaCTX-M-1 group + blaIMP
blaCTX-M-9 group only
blaCTX-M-9 group + blaCMY-2 -like
blaCTX-M-2 groupa
blaSHV-5/12 -type only
blaTEM-12 b
126
6
5
3
1
47
1
1
17
1
67
4
0
1
0
44
1
0
5
1
59
2
5
2
1
3
0
1
12
0
MBL (n = 22)
blaIMP only
blaIMP + blaDHA
20
1
2
0
18
1
ampC (n = 34)c
blaCMY-2 -like only
blaDHA only
blaMIR
23
5
1
19
0
0
4
5
1
258/267
144/152
114/115
Other ␤-lactam
resistance
blaTEM
blaOXA-30 -like
blaOXA-23 -like
152d
101
1
89
44
1
63
57
0
Aminoglycoside
resistance
aac(3)-II
aac(6 )e
aadB
155
30
7
70
5
3
85
25
4
PMQR
aacA4cr
qnrA
qnrB
qnrS
88
2
49
8
39
1
1
2
49
1
48
6
Explained/total
ESBL, extended-spectrum ␤-lactamase; MBL, metallo-␤-lactamase; PMQR, plasmid-mediated quinolone resistance.
a
Identified as blaCTX-M-2 group by additional PCR and sequencing.
b
The blaTEM gene detected by the multiplex PCR/reverse line blot (mPCR/RLB) assay was sequenced in this case because no other genes to explain the ESBL phenotype
were identified.
c
blaMIR gene identified by additional multiplex PCR. Six additional E. coli with no explanatory gene(s) had altered AmpC promoter sequences (not shown here, see Table 4).
d
This includes one isolate with blaTEM-12 and one with blaTEM-30 (Table 4). No other blaTEM genes were sequenced.
e
Contain an aacA4 subgroup gene that is not an aacA4cr variant.
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cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents (2013),
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in 207 (77.5%) of the 267 clinical isolates of E. coli (n = 152) and
K. pneumoniae (n = 115). Additional PCR identified these as
blaCTX-M-1 group genes (141/207; 68.1%) and blaCTX-M-9 group genes
(54/207; 26.1%), confirmed the presence of blaSHV-5/12 -type genes
in 22/207 (10.6%) of the isolates and demonstrated the absence of
blaVEB from all isolates. A blaCTX-M gene in one isolate was identified as blaCTX-M-2 -like by PCR and partial sequencing. Of these
207 isolates, 11 (5.3%) had two ESBL genes (Table 3). Several isolates (22/267; 8.2%), mostly K. pneumoniae (20/115; 17.4%), had a
blaIMP gene (Table 3), including one with a co-existing ESBL gene
(blaCTX-M-1 group). Plasmid-borne ampC genes were detected by
mPCR/RLB assay in 33/267 isolates (12.4%). blaCMY-2 -like genes
were more common in E. coli (n = 21, 2 of which also had a blaCTX-M
gene) than K. pneumoniae (n = 6, 2 of which also had a blaCTX-M gene).
In contrast, blaDHA -like genes were only detected in K. pneumoniae (n = 6, 1 of which also had blaIMP ). blaTEM genes were detected
in 152/267 isolates (56.9%), but as ESBL-type TEM appears to be
rare locally and blaTEM-1 was commonly found in isolates with ESBL
genes [6], these genes were only characterised in selected isolates
(see Section 3.2). No blaOXA-10 -like genes were detected.
3.2. Detection of additional genes that could explain
non-susceptibility to cefotaxime or ceftazidime
Of the 267 isolates, 11 (4.1%) had no explanation for nonsusceptibility to third-generation cephalosporins identified by
first-pass screening by this mPCR/RLB assay if blaTEM is discounted
(Table 4). Multiplex PCR for additional plasmid-borne ampC genes
not present in the mPCR/RLB assay revealed a blaMIR gene in a single
K. pneumoniae (NEP KP09) with an AmpC phenotype, and sequencing of blaTEM in the three E. coli where this was the only ␤-lactamase
gene detected revealed an ESBL TEM gene, blaTEM-12 [22] in one
isolate with a modest ESBL phenotype (CON EC05).
Sequencing of the chromosomal ampC promoter regions
revealed alterations (not shown) that might cause chromosomal
AmpC hyperproduction [29] in all six E. coli with still unexplained
AmpC phenotypes, including CON EC08 with blaTEM-1b (encoding
a penicillinase) and SVH EC12 with blaOXA-30 (cefepimase). Details
are outside the scope of this study, but two of these six were clearly
CTX-susceptible and only borderline CAZ-non-susceptible; only
one (CON EC21) had MICs well beyond the resistance breakpoints
(Table 4).
No explanatory gene was identified for a weak ESBL phenotype
in E. coli RPA EC19, nor for a slightly elevated CAZ MIC in K. pneumoniae CON KP30 (Table 4). Escherichia coli CON EC04, with only
an inhibitor-resistant TEM-30, had borderline susceptible CTX and
CAZ MICs.
3.3. Detection of associated aminoglycoside resistance genes
Of the 267 isolates, 175 were non-susceptible (MIC ≥ 8 ␮g/mL)
to gentamicin. At least one potentially causal gene was detected
in 170 of these by mPCR/RLB assay [aac(3)-II, n = 133; aac(3)-II
plus aacA4 subgroup, n = 22; aacA4 subgroup, n = 8; aadB, n = 7].
aacC1 was not detected in any isolates. None of the 92 gentamicinsusceptible isolates had any of the aminoglycoside resistance genes
tested for. The phenotypes of the remaining five isolates [four
resistant to gentamicin (MIC > 8 ␮g/mL) and one with intermediate resistance (MIC = 8 ␮g/mL)] could not be explained by any of
the genes screened for. Thus, 97% of the gentamicin resistance
associated with resistance to third-generation cephalosporins
could be explained by three gene types. Only four isolates were
non-susceptible to amikacin and this resistance was largely unexplained. Neither armA nor rmtC were detected by the mPCR/RLB
assay. As no isolates were resistant to amikacin, gentamicin and
tobramycin, characteristic of 16S rRNA methylases, other methylase genes were not screened for.
3.4. Detection of associated plasmid-mediated quinolone
resistance determinants
The aacA4cr variant was detected in 49/115 (42.6%) of K. pneumoniae isolates and 49/152 (32.2%) of E. coli. qnrA and qnrS genes
were rarely detected, with a combined total of 10/267 (3.7%), whilst
qnrB genes were relatively common in K. pneumoniae (48/115;
41.7%) but not in E. coli (1/152; 0.7%).
3.5. Combinations of resistance genes found in known
multiresistance regions
Part of the reason that some resistance genes appear more common than others is likely to be their association with successful
plasmids and/or with other resistance genes, which may allow
co-selection by different antibiotics, in multiresistance regions
(MRRs). The blaCTX-M-15 gene, which belongs to the blaCTX-M-1 group,
has been found on IncF-type plasmids within large MRRs that also
include aac(3)-IIe, the |aacA4cr|blaOXA-30 |catB3 cassette array and
blaTEM-1b , or variants of this MRR with one or more of these genes
missing [15]. Of the 141 isolates with a blaCTX-M-1 group gene, 30
(21.3%) also had aac(3)-II, aacA4cr, blaOXA-30 -like and blaTEM genes,
whilst 42/141 (29.8%) had the first three genes but lacked blaTEM ,
suggesting the presence of this type of MRR.
Similarly, the blaIMP-4 gene is common in Sydney [7], where it is
typically found on an IncL/M plasmid that also carries the aac(3)IId gene, an aacA4 gene (not the cr variant) and qnrB2 (GenBank
accession no. JX101693). Of 22 isolates with a blaIMP gene, 19 had
aac(3)-II, aacA4 subgroup and qnrB genes, consistent with the presence of this plasmid.
4. Discussion
We show here that a limited set of targets can be used to predict third-generation cephalosporin resistance in Sydney, Australia.
A relevant gene was identified in nearly every isolate with MICs of
CTX or CAZ beyond resistance MIC breakpoints. At least one target,
representing only seven gene types (blaCTX-M , blaSHV-5/12 , blaIMP ,
blaCMY-2 -like, blaDHA , blaMIR -like and blaTEM ) was identified that
could explain non-susceptibility in 96% of the isolates tested, being
99% (114/115) of K. pneumoniae and 93% of E. coli (142/152). Only
two of the non-susceptible isolates (CTX/CAZ MIC ≥ 2 ␮g/mL) that
included none of these few targets were very resistant (Table 4)
and only one was well beyond the CTX resistance breakpoint (NEP
K09, which had a plasmid AmpC gene not included in the mPCR/RLB
screening targets). Only 2 of 267 non-susceptible isolates remain
truly unexplained (CON KP30, CAZ MIC = 3 ␮g/mL; and RPA EC19,
CAZ MIC = 6 ␮g/mL).
At least one relevant target (of only three gene types represented) that could explain the associated non-susceptibility to
gentamicin was also detected in 97% of these isolates, being 98%
(94/96) of K. pneumoniae and 96% of E. coli (76/79). Inclusion of additional gene targets, identified by surveillance, should increase this
accuracy, but the extent to which the law of diminishing returns
operates is unknown. Addition of gentamicin resistance genes into
the prediction algorithm for third-generation cephalosporin resistance would further increase sensitivity, since 2 of the 11 isolates
needing further investigation also had a gentamicin resistance gene
detected. Our local ESBL gene pool appeared relatively stable over
3 years [5,6], but target sets may need to be periodically revised to
include additional/different markers (e.g. during an outbreak of a
previously rare gene). Ongoing surveillance will clearly be needed
to determine how often such modifications are required.
Please cite this article in press as: Ginn AN, et al. Limited diversity in the gene pool allows prediction of third-generation
cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents (2013),
http://dx.doi.org/10.1016/j.ijantimicag.2013.03.003
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7
Table 4
Isolates further investigated after multiplex PCR/reverse line blot (mPCR/RLB) assay.
Isolate
Klebsiella pneumoniae
NEP KP09
CON KP30
Escherichia coli
CON EC04
CON EC05
RPA EC19
CON EC08
CON EC21
SVH EC12
SVH EC15
WM EC12
RPA EC03
Genes detecteda
Phenotype
blaMIR
AmpC
–
blaTEM-30 d ; aac(3)-II
blaTEM-12 d
–
blaTEM-1b ; aac(3)-II
–
blaOXA-30 f
–
–
–
–
ESBL
ESBL
AmpCe
AmpCe
AmpCe
AmpCe
AmpCe
AmpCe
MIC (␮g/mL)
CTXb
CAZb
>16
0.5
>16
3
2
1
2
8
6
4
24
8
8
2
3
≤0.5
≤0.5
≤0.5
1
≤0.5
>16
≤0.5
≤0.5
≤0.5
0.75
2
0.25
1.5
8
2
4
0.75
0.5
FEPc
MIC, minimum inhibitory concentration; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; EUCAST, European Committee on Antimicrobial Susceptibility Testing; CLSI,
Clinical and Laboratory Standards Institute; ESBL, extended-spectrum ␤-lactamase.
a
All blaTEM genes in Table 4 were detected by mPCR/RLB assay before PCR and sequencing.
b
MICs for CTX and CAZ by Etest (NEP KP09 by Phoenix). Susceptibility and resistance breakpoints, respectively: EUCAST: CTX, 1 ␮g/mL and 2 ␮g/mL; CAZ, 1 ␮g/mL and
8 ␮g/mL; CLSI: CTX, 1 ␮g/mL and 4 ␮g/mL; CAZ, 4 ␮g/mL and 16 ␮g/mL.
c
FEP MICs by Phoenix. Susceptibility and resistance breakpoints, respectively: EUCAST: 1 ␮g/mL and 4 ␮g/mL; CLSI: 4 ␮g/mL and 8 ␮g/mL.
d
TEM-30 is an ‘inhibitor-resistant TEM’; TEM-12 is a known ‘ESBL TEM’.
e
Altered chromosomal AmpC promoter sequences detected (data not shown).
f
OXA-30 is known to hydrolyse FEP.
Table 5
Estimated negative predictive values (%) for non-susceptibility of the multiplex PCR/reverse line blot (mPCR/RLB) assay at different background prevalence rates.
Prevalence rate (%)
5
10
50
67
80
Third-generation cephalosporin-resistant
Gentamicin-resistant
Escherichia coli
Klebsiella pneumoniae
Combined
Combined
99.65
99.27
93.83
88.22
79.17
99.95
99.90
99.14
98.27
96.64
99.78
99.54
96.04
92.28
85.85
99.85
99.68
97.22
94.52
89.74
An ESBL blaTEM-12 gene was identified here, but including all
ESBL blaTEM variants [22] is impractical and probably unnecessary. Unlike SHV, where most mutations giving an ESBL phenotype
occur in adjacent codons that can be targeted by a single probe,
mutations conferring an ESBL phenotype on TEM are more widely
spread throughout the protein [22]. Certain ESBL blaTEM variants
are known to be prevalent in some countries, and including these
in locally targeted assays may be necessary to achieve highly predictive tests for the third-generation cephalosporins.
This study was not designed as a survey but as a proofof-principle. We chose targets most commonly associated with
the phenotype of interest, typically plasmid-encoded and readily
mobilised between species under selection pressure, some associated with a MIC within potentially achievable tissue levels of a
given antibiotic but nevertheless associated with increased risk of
treatment failure. We relied primarily on a probe-based method
that we also describe herein, but this principle applies to targeting
with any nucleic acid-based detection platform with multiplexing
capacity and some flexibility in target recognition at the level of
stringency variation and/or probe/primer redundancy. The ultimate aim would be to use a more limited number of predictive
targets, as identified here, to develop rapid assays for use with
clinical specimens.
The NPV is the most useful measure of the confidence with
which antibiotic efficacy can be predicted by this approach and
is inversely related to background prevalence (pre-test probability). In countries such as Australia, with a pre-test probability
of resistance to the common third-generation cephalosporins of
5–10% in E. coli and K. pneumoniae, the NPV of >99.5% would suffice
to guide empirical antibiotic choice in septic shock [30]. As the
background resistance rate (and pre-test probability) rises, the
NPV falls (Table 5). This is true even if target diversity does not
increase, although the extent to which that also occurs in countries
with more resistance is an important unknown to address.
Acknowledgments
The authors thank Elaine Cheong (Concord Hospital, Sydney, Australia) for constructive advice and support; Fanrong
Kong (Westmead Hospital, Sydney, Australia) for advice on the
mPCR/RLB assay; and Jan Bell and John Turnidge (SA Pathology,
Adelaide, Australia) for some control isolates.
Funding: Funding support was received from the National Health
and Medical Research Council of Australia (grants 512396 and
1001021).
Competing interests: None declared.
Ethical approval: Not required.
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Please cite this article in press as: Ginn AN, et al. Limited diversity in the gene pool allows prediction of third-generation
cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents (2013),
http://dx.doi.org/10.1016/j.ijantimicag.2013.03.003