Genetic and Biochemical Characterization of a Novel Metallo-
?-Lactamase, TMB-1, from an Achromobacter xylosoxidans Strain
Isolated in Tripoli, Libya
Allaaeddin El Salabi,aPardha Saradhi Borra,dMark A. Toleman,aØrjan Samuelsen,band Timothy R. Walsha,c
Cardiff University, Cardiff, United Kingdoma; Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University
Hospital of North Norway, Tromsø, Norwayb; University of Queensland, Centre for Clinical Research, Queensland, Australiac; and Research Group for Host-Microbe
Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norwayd
An Achromobacter xylosoxidans strain from the Tripoli central hospital produced a unique metallo-?-lactamase, designated
mosome.TheTMB-1 ?-lactamasehaslower kcatvaluesthanbothDIM-1andGIM-1withcephalosporinsandcarbapenems.The
ment of Gram-negative infections worldwide, such that most
MBL-producing organisms are only susceptible to colistin (2).
These enzymes very efficiently hydrolyze all ?-lactams, including
carbapenems (with the exception of aztreonam), and the ?-lacta-
mase genes most often are located on transferable genetic plat-
forms, namely, either ISCR elements or class 1 integrons some-
times embedded in Tn21- or Tn402-like transposons (22, 24).
However, several recently characterized MBL genes have been
flanked or associated with ISCR elements, namely, blaSPM-1with
ISCR4, blaNDM-1with ISCR1, and blaAIM-1with ISCR16 (6, 21).
NDM, IMP, and VIM derivatives being the most widespread (2).
blaNDM-1has mainly been found in Enterobacteriaceae (2, 6, 11,
ical locations, including SIM-1 from Acinetobacter baumannii in
Korea (8) and KHM-1 from Citrobacter freundii in Japan (16).
SPM-1 in Brazil (10, 23), GIM-1 in Germany (3), and AIM-1 in
Australia (T. R. Walsh, unpublished data) were all identified in
ative bacteria, and because in Libya adherence to internationally
accepted infection control policies is not optimal, we examined
tance to extended-spectrum cephalosporins. This study reports
these findings and further describes the genetic and biochemical
characterization of a novel MBL, TMB-1 (for Tripoli metallo-?-
lactamase), from Tripoli, Libya.
obile metallo-?-lactamases (MBLs) are becoming increas-
ingly frequent and pose significant challenges to the treat-
MATERIALS AND METHODS
Bacterial strains and susceptibility testing. A total of 38 nonclinical en-
vironmental swabs (from hospital wards, cafes, corridors, ventilators,
floors, bedside cabinets, oxygen cylinders, electrocardiograph machines,
transferring charcoal media, and bacteria were selected by culturing on
MacConkey agar (Oxoid, United Kingdom) supplemented with 100 mg/
2 mg/liter of ceftazidime to select for strains resistant to extended-spec-
trum cephalosporins. Isolates were initially identified by the use of Phoe-
Plane, France). The susceptibility tests were performed by Phoenix 100
(Becton Dickinson, Oxford, United Kingdom) and Etest strips (bioMéri-
eux, La Plane, France) and were interpreted by the European Committee
on Antimicrobial Susceptibility Testing (http://www.eucast.org/eucast
Phenotypic and molecular detection of MBLs. Phenotypic MBL de-
a substrate (bioMérieux, La Plane, France), and the results were inter-
preted according to the manufacturer’s instructions.
Identification of blaTMB-1, PCR experiments, and cloning. Molecu-
lar screening was performed on all isolates targeting the conserved region
of class 1 integrons, ISCR elements, and Tn21- and Tn402-like trans-
posons (Table 1). The PCR conditions were undertaken as previously
of class 1 integrons were run on 1% (wt/vol) agarose gels, purified, and
Ltd., Paisley, United Kingdom) and sequenced using the primers listed in
Table 1. All of the PCR products were run on 1% (wt/vol) agarose gel,
purified, and sequenced using an automated sequencer (AB 377; Perkin-
Conjugation experiments. The conjugational transfer of antibiotic
resistance to the laboratory strain E. coli J53 (azide resistant) and Pseu-
domonas aeruginosa PAO1 (rifampin resistant) was carried out on blood
agar without selection. After 18 h, the mixed cultures were washed from
ing sodium azide (100 ?g/ml) and ceftazidime (2 ?g/ml) or rifampin (50
?g/ml) and ceftazidime (2 ?g/ml). Ceftazidime-resistant colonies were
screened for blaTMB-1by the primers listed in Table 1.
Hybridization. Gel plugs of chromosomal DNA were prepared and
restricted with S1 nuclease as previously described (27). Hybridization
Received 31 August 2011 Returned for modification 6 November 2011
Accepted 15 January 2012
Published ahead of print 30 January 2012
Address correspondence to Timothy R. Walsh, WalshTR@Cardiff.ac.uk.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0066-4804/12/$12.00Antimicrobial Agents and Chemotherapyp. 2241–2245aac.asm.org
was performed in a gel. Briefly, the gel was dried for 5 h at 50°C and then
in a denaturing solution (0.5 M NaOH, 1.5 M NaCl) and neutralizing
gel was then prehybridized at 65°C using prehybridizing solution (20 ml;
6? SSC [1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1%
[wt/vol] polyvinylpyrrolidone 400, 0.1% [wt/vol] Ficoll, 0.1% [wt/vol]
bovine serum albumin [BSA; Cohn fraction V], 0.5% [wt/vol] SDS, and
150 ?g/ml denatured calf thymus DNA) for at least 4 h before adding the
probe. The32P-labeled probe was added for a minimum of 8 h at 65°C
by 0.1? SSC, 0.1% SDS for 3 h; both washes were performed at 65°C.
blaTMB-1probes were labeled with32P by using a random primer tech-
nique (Stratagene, La Jolla, CA).
Purification of TMB-1. To negate posttranslational modification in
an unnatural host, TMB-1 was purified directly from strain AES301,
which was grown overnight at 37°C in Terrific broth (Sigma, St. Louis,
MO). TMB-1 was purified from the periplasm (15), followed by ion ex-
change (Q-Sepharose HP column; Pharmacia, GE Healthcare, United
50 mM Tris (pH 7.2), 100 ?M ZnCl2, 0.02% (wt/vol) sodium azide, with
or without NaCl, as the buffer system. Fractions containing TMB-1 were
FIG 1 Genetic context of two class 1 integrons found in A. xylosoxidans and the primers used to sequence the structures. (A) Class 1 integron consisting of the
gene cassettes containing dhfrA4, aacA4, blaOXA-4, and the qacE?/sulL fusion. (B) Class 1 integron consisting of the gene cassettes containing blaTMB-1, aacA4,
blaOXA-4, and qacE?/sulL. The white ellipses represent the hybrid promoter from intI1. The black ellipses represent the 59-bp elements at the start of each gene
cassette. The arrows depict primers used to amplify and sequence the integrons, and the sequences are given in Table 1.
TABLE 1 Primers used in this study
Gene targetPrimer no.a
Primer namePrimer sequence Reference or source
Quaternary ammonium compound
GCC TGT TCG GTT CGT AAG CT
CGG ATG TTG CGA TTA CTT CG
GCC AAC GAA GAA ATA CCC GC
TGG GCT AGG TTA CAC TGG TG
TTC TAG CGG ATT GTG GCC AC
CAA GGA GCT CAT TCA AAGG
GGA GCA GGC AAG GAG CT
ACC CGG ATT GGA AGT TGA GG
TGA TCA GTG GCC ACA ATC CG
CGG ATT GTG GCC ACT GAT CA
CGA AGA ATG GAG TTA TCG GG
GTT AGA GGC GAA GTC TTG GG
CCC AAG ACT TCG CCT CTA AC
GGC GTC GGC TTG AAT GAG TT
AAG TGG CAG CAA CGG ATT CG
GAA TCC GTT GCT GCC ACT TG
CAA CTC ATT CAA GCC GAC GC
CAC TTA TGG CAT TTG ATG CG
CGC ATC AAA TGC CAT AAG TG
GGG GTC GTC TCA GAA AAC GG
GGA AAA TAA AGC ACG CTA AG
GTC GTT TTC AGA AGA CGG C
CTA TGC TCA ATA CTC GTG TGC
GGT TGC AAC GAC TCA AGCG
CAC TCG TTT ACC GCT CAA GC
aThe primer numbers are the same as those used in Fig. 1.
El Salabi et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
FIG2 (A) Comparison of amino acid sequence of the ?-lactamase TMB-1 to those of other acquired MBLs (DIM-1, GIM-1, IMP-1, KMH-1, NDM-1, VIM-1,
SPM-1, and SIM-1) and several naturally occurring MBLs (IND-1 from Chryseobacterium indologenes, JOHN-1 from Flavobacterium johnsoniae, SLB-1 from
Shewanella livingstonensis, and SFB-1 from Shewanella frigidimarina). Shaded amino acids are those conserved with TMB-1. ?-Lactamase numbering was
according to the BBL nomenclature (5). (B) Secondary structure of TMB-1 compared to that of VIM-2. The ?-strands and ?-helixes are indicated above the
TMB-1 sequence. The conserved residues are indicated in black. The conservative amino acid substitutions are boxed. The figure was obtained using ESPript
A. xylosoxidans Metallo-?-Lactamase TMB-1 from Libya
May 2012 Volume 56 Number 5 aac.asm.org 2243
analyzed using nitrocefin with or without EDTA, and the fractions were
analyzed by SDS-PAGE (Invitrogen, CA). TMB-1 was concentrated to
1.94 mg/ml using ultrafiltration (Millipore, MA).
Kinetics assays. Steady-state kinetics were performed at 25°C in a
spectrophotometer (SpectramaxPlus; Molecular Devices) using 96-well
plates (BD Falcon UV microplates; BD Biosciences) (15). All substrates
were tested as duplicates using 50 mM HEPES buffer, pH 7.2, 100 ?M
ZnCl2, 0.02% NaN3, and 0.1 mg/ml bovine serum albumin (Sigma-Al-
drich) as a buffer system. The kinetic data were analyzed by nonlinear
regression (GraphPad Software, San Diego, CA).
Nucleotide sequence accession number. The full sequence of the
3-kb class 1 integron reported in the present study has been submitted to
the EMBL/GenBank and assigned nucleotide sequence accession number
RESULTS AND DISCUSSION
were positive for class 1 integrons: one Achromobacter xylosoxidans
isolate (two integrons of ?3 kb), one Stenotrophomonas maltophilia
lowing MIC profile: imipenem, 2 ?g/ml; meropenem, 4 ?g/ml;
treonam, 16 ?g/ml; amikacin, 8 ?g/ml; gentamicin, 8 ?g/ml; cipro-
floxacin, 1 ?g/ml; and colistin, 0.5 ?g/ml. All isolates that grew on
detect the presence of MBLs. Apart from the S. maltophilia isolate
itive isolate was an A. xylosoxidans strain, designated AES301, pos-
strain AES301. The sequencing analysis of the class 1 integron
PCR products from A. xylosoxidans AES301 revealed two nearly
identical integrons, the first possessing the gene cassette dhfrA4-
aacA4-blaOXA-4and the second integron containing the gene cas-
could not be mated to either E. coli DH5? or P. aeruginosa PAO1
is chromosomally located. This inference was supported by
Southern hybridization data using the blaTMB-1gene as a probe,
which was back blotted to the A. xylosoxidans AES301 chromo-
The TMB-1 gene contains 735 nucleotides and encodes a pro-
tein of 245 amino acids possessing all of the key motifs of Ambler
class B ?-lactamase. At the amino acid level, TMB-1 was most
closely related to DIM-1 (62%) and GIM-1 (51%) and showed
only 48, 31, and 29% identity to IMP-1, VIM-2, and NDM-1,
secondary residues supporting the active sites, including the pu-
tative loop used to facilitate the binding of ?-lactams during hy-
drolysis (Fig. 2A). A secondary structural comparison of TMB-1
to VIM-2 shows that TMB-1 possesses the key zinc binding resi-
dues for B1 MBLs, namely, His116, His118, and His196 (zinc 1)
and Asp120, Cys221, and His263 (zinc 2) (Fig. 2A). The most
terminus of the TMB-1 protein just before the beginning of the
first ?-sheet (?1 in Fig. 2B). This gap in TMB-1 is situated just
prior to the flapping loop of VIM-2, which has been shown to
facilitate the binding and hydrolysis of substrates (1). Further,
there are several amino acid differences in this region, namely
(comparing VIM-2 to TMB-1), Q60S, S61R, F62V, D63E, A66G,
to a more flexible flapping loop (1). Interestingly, DIM-1 pos-
sesses the same sequence as TMB-1 in this region, with the excep-
tion of the gap and the amino acid changes N63E and F65W (12
?8 compared to the VIM-2 sequence is also observed (Fig. 2B).
were compared to those of DIM-1 and GIM-1 (Table 2) and are
broadly similar, with the exception of the rate of turnover of sub-
to those of DIM-1 and GIM-1 for the penicillins and cephalospo-
rins but are larger for meropenem, indicating that TMB-1 binds
meropenem comparatively weakly. The kcatvalues for TMB-1 are
similar between the pencillins and GIM-1 but are markedly less
(30- to 260-fold) than those for both DIM-1 and GIM-1 for ce-
TABLE 2 Steady-state kinetic constants of TMB-1, DIM-1, and GIM-1
Steady-state kinetic constants ofc:
aData are from Poirel et al. (12).
bData are from Castanheira et al. (3).
cNR, not reported. ND, not detected.
El Salabi et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
foxitin, cefuroxime, and ceftazidime (Table 2). TMB-1 also pos- Download full-text
sesses lower kcatvalues for the carbapenems (2- to 36-fold) com-
are interesting, given that TMB-1 and DIM-1 are similar and that
their sequences in the VIM-2 flapping loop (15) are nearly iden-
tical, further suggesting that the reasons for these kinetic differ-
ences lie elsewhere in the TMB-1 structure (Fig. 2B).
growing number of reports indicate that it is capable of causing
urinary tract infections (20), ocular infections (13), and the con-
tamination of dialysis (25) and ultrasound equipment (9), and it
can cause additional complications in cystic fibrosis patients (7,
a ward surface swab, the same strain could not be identified from
a clinical source; however, in Libya clinical diagnostic microbiol-
ogy laboratories may not scrutinize strains to the species level. To
date, only two cases of MBL genes (both blaVIM-2) have been re-
ported from Achromobacter spp., from Greece (19) and Korea
(18), and both were carried by class 1 integrons. This is the first
MBL reported from Libya, and being a new subclass, it provides
further evidence of the structural heterogeneity of this group of
A.E.S. was supported by an overseas scholarship from the Libyan govern-
ment. Project money for the work in Cardiff was provided by the Euro-
Northern Norway Regional Health Authority.
Janis Weeks provided technical assistance at Cardiff University.
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