International Journal of Antimicrobial Agents 25 (2005) 157–162

Evolution of CTX-M-type ␤-lactamases in isolates of Escherichia coli

infecting hospital and community patients
Gioconda Brigantea , Francesco Luzzaroa , Mariagrazia Perillib , Gianluigi Lombardia ,
Alessandra Colı̀a , Gian Maria Rossolinic , Gianfranco Amicosanteb , Antonio Tonioloa,∗
a Laboratory of Medical Microbiology, Ospedale di Circolo and University of Insubria, Viale Borri 57, 21100 Varese, Italy
b Department of Sciences and Biomedical Technology, University of L’Aquila, L’Aquila, Italy
c Department of Molecular Biology, University of Siena, Siena, Italy

Received 2 August 2004; accepted 14 September 2004

Abstract

Escherichia coli isolates collected at our Institution from 1999 to 2003 (n = 20,258) were studied to evaluate the production of CTX-M-type
extended-spectrum ␤-lactamases (ESBL). Isolates suspected of producing CTX-M enzymes were analyzed by the double-disk synergy test,
hybridization with specific probes, PCR and direct DNA sequencing. Overall, 53 ESBL-positive isolates were found to carry CTX-M-type
genes (blaCTX-M-1 , n = 51; blaCTX-M-15 , n = 2). The isolation of CTX-M-positive strains increased from 1 per year (1999) to 26 per year (2003).
The first isolate carrying the blaCTX-M-15 gene appeared in 2003 and was obtained from a patient previously treated with ceftazidime. CTX-M-
positive isolates were characterized by multi-drug resistance and were obtained both from inpatients (n = 29) and outpatients (n = 24). Most
patients were over 60-year-old (n = 45), had underlying chronic diseases (n = 32), and had been hospitalized more than once (n = 33). Strains
were frequently isolated from the urinary tract, often after recurrent infections. Our study demonstrates that CTX-M-producing isolates are
increasing among E. coli strains. Adequate laboratory detection may help in choosing appropriate treatment and in limiting the spread of this
resistance trait.
© 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Keywords: ESBL; Cefotaximases; Multi-drug resistance; Urinary tract infection

1. Introduction tected CTX-M enzyme (M-1 type) was classified as ce-

fotaximase since it showed higher levels of hydrolytic
Expression of extended-spectrum ␤-lactamases (ESBL) activity against cefotaxime than against ceftazidime (at
in enterobacteria is of great clinical relevance [1]. These least 35-fold difference level). However, some recently de-
enzymes are commonly derived from TEM-1/2 or SHV- scribed CTX-M enzymes (i.e., CTX-M-15, CTX-M-27 and
1 ␤-lactamases by a limited number of mutations that en- CTX-M-16, which are derived by a Gly240Asp substitu-
able ESBL-positive bacteria to hydrolyze all penicillins, tion from CTX-M-3, CTX-M-9 and CTX-M-14, respec-
cephalosporins and aztreonam [2]. tively) have greater catalytic activity against ceftazidime
Enzymes of the CTX-M-type are non-TEM- and [4–6]. It was soon realized that CTX-M-type genes were
non-SHV-derived ESBL, which have been increasingly characterized by a great ability to spread [7,8]. CTX-M-
reported world-wide over the last decade [3]. The CTX- producing Escherichia coli isolates have been detected in
M family comprises approximately 40 different enzymes different geographical areas. Most reported enzymes are
[3, http://www.lahey.org/studies/webt.htm]. The first de- of the CTX-M-1 group (e.g., M-1, M-3, M-10 and M-15
types), but enzymes of the CTX-M-9 group (e.g., M-9, M-
∗
14, M-16, M-21 and M-27 types) have also been reported
Corresponding author. Tel.: +39 0332 278309; fax: +39 0332 260517.
E-mail address: antonio.toniolo@ospedale.varese.it (A. Toniolo).
[3].

0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/j.ijantimicag.2004.09.013
158 G. Brigante et al. / International Journal of Antimicrobial Agents 25 (2005) 157–162

Data on the prevalence and distribution of different CTX- tion were performed on nylon membranes (Hybond-N,
M-type enzymes are very limited. Some European reports Amersham-Pharmacia Biotech, Milan, Italy) according to
suggest that CTX-M-type enzymes may account for up to the manufacturer’s instructions, using random-primed 32 P-
50% of ESBL-positive strains in E. coli isolates [9,10]. An labelled DNA probes. PCR-generated amplicons contain-
Italian nation wide survey conducted in 1999 did not de- ing either the blaTEM-1 or the blaSHV-1 genes were used as
tect CTX-M enzymes in clinical enterobacterial isolates [11]. probes to detect the presence of blaTEM and blaSHV alle-
More recently, CTX-M enzymes have been reported from les, respectively. The presence of CTX-M-type genes was
some Italian laboratories, the CTX-M-1 enzyme being the assessed using specific DNA probes designed on conserved
only one detected in E. coli [12,13]. regions of blaCTX-M genes. PCR amplification of blaCTX-M
This study was initiated to investigate the prevalence of alleles was carried out with the following primers: CTX-
CTX-M-type enzymes in community- and hospital-acquired F (5 -ATGGTTAAAAAATCACTGCGCCAG) and CTX-R
isolates of E. coli collected at our Institution (Varese, North- (5 -CAAACCGTCGGTGACGATTTTAG). PCR conditions
ern Italy) over a 5-year period (1999–2003). Phenotypic and were as follows: denaturation at 94 ◦ C for 30 s, annealing at
molecular methods (hybridization with DNA probes, PCR 56 ◦ C for 1 min, extension at 72 ◦ C for 1 min, repeated for
and direct sequencing) were used to characterize CTX-M de- 30 cycles and a final extension at 72 ◦ C for 7 min. Direct se-
terminants detected in E. coli isolates. quencing of PCR amplicons was performed on both strands
by the dideoxy-chain termination method using a dRho-
damine Terminator Cycle Sequencing Ready Reaction Kit
2. Materials and methods and the ABI PRISM 377 DNA Sequencer (Applied Biosys-
tem) and custom sequencing primers.
2.1. Clinical isolates and antimicrobial susceptibility E. coli isolates were genotyped by an enterobacte-
testing rial repetitive intergenic consensus-PCR method using
25 pM of the ERIC2b primer 5 -AAGTAAGTGACT-
Non-repetitive consecutive isolates of E. coli obtained GGGGTGAGCG-3 [16]. The reaction was carried out in
from clinical specimens at the Microbiology Laboratory of 50 ␮l as described with minor modifications: MgCl2 4 mM,
Ospedale di Circolo e Fondazione Macchi (Varese, Italy) over denaturation at 94 ◦ C for 30 sec, annealing at 55 ◦ C for 1 min,
a 5-year period (1999–2003) were studied. Both inpatients extension at 72 ◦ C for 1 min, repeated for 34 cycles. Profiles
and outpatients were included in the study. Bacterial identifi- were assessed by visual inspection of images of ethidium
cation and susceptibility testing were performed using broth bromide-stained agarose gels containing molecular size stan-
microdilution panels (Sceptor and Phoenix systems; Becton dards. Determinations were done in duplicate and included
Dickinson Diagnostic Systems, Sparks, MD). Isolates that negative controls.
conformed to the criteria of the National Committee for Clin-
ical Laboratory Standards [14] and that were suspected of 2.3. Clinical data
producing ESBL were further investigated. The double-disk
synergy method was used to confirm ESBL production [15]. Clinical records of hospitalized patients infected by CTX-
The test was performed as a standard disk diffusion assay on M-producing E. coli were examined retrospectively to assess
Mueller-Hinton agar (Oxoid, Milan, Italy). Disks containing associated risk factors and underlying clinical conditions. The
30 ␮g of aztreonam, cefotaxime, ceftazidime, ceftriaxone, following data were evaluated: age, gender, microbiologi-
cefepime and cefpodoxime were placed 25 mm apart (cen- cal specimen, clinical ward and hospitalization during the
tre to centre) around a disk containing amoxicillin (20 ␮g) 1-year period before infection, use of cephalosporins during
plus clavulanic acid (10 ␮g). Enhancement of the inhibition the 2-month period before infection, diagnosis at admission,
zone, indicating a synergy between clavulanic acid and any severity of the underlying disease according to the Charlson
one of the test antibiotics was considered suggestive of ESBL weighted index [17], repeated isolation of the same micro-
production. The synergistic activity of clavulanate with both organism.
cefotaxime and ceftazidime was confirmed by means of two Outpatients investigations were limited to demographic
Etest special strips (AB Biodisk, Solna, Sweden) containing data, microbiological specimen, previous hospitalization, use
either drug alone or in combination with clavulanate. A ≥3 of antibiotics and repeated isolation of the same micro-
two-fold dilution decrease in the MIC of either cefotaxime organism.
or ceftazidime was considered positive according to NCCLS
guidelines [14].
3. Results
2.2. Molecular methods
A total of 20,258 consecutive, non-repeated E. coli
ESBL determinants of the TEM-, SHV- and CTX-M- isolates were investigated at our Institution over a 5-year
types were investigated by colony-blot hybridization and period. Of these, 16,834 (83.1%) were obtained from the
PCR. Colony-blot hybridization and Southern blot hybridiza- urinary tract (10,607 from outpatients, 6227 from inpatients)
 G. Brigante et al. / International Journal of Antimicrobial Agents 25 (2005) 157–162 159

Table 1
Increasing isolation rate of CTX-M-positive E. coli strains
Year No. of studied isolates No. of ESBL-positive No. of CTX-M-positive No. of isolates carrying
isolates (%)a isolates (%)b different CTX-M genes
blaCTX-M-1 blaCTX-M-15
1999 3600 8 (0.22) 1 (12.5) 1 0
2000 3991 31 (0.78) 7 (22.6) 7 0
2001 4374 43 (1.00) 9 (20.9) 9 0
2002 4021 50 (1.24) 10 (20.0) 10 0
2003 4272 68 (1.59) 26 (38.2) 24 2
Total 20,258 200 (0.99) 53 (26.5) 51 2
a Percent of ESBL-positive isolates with regard to the number of isolates.
b Percent of CTX-M-positive isolates with regard to the number of ESBL-positive isolates.

and 3424 (16.9%) from non-urinary infections (1948 from against this drug). The response to cefotaxime and ceftriax-
outpatients, 1476 from inpatients). Overall, 200 isolates one was also strongly reduced.
(approximately 1%) were found to produce an ESBL Table 2 shows the ␤-lactam susceptibility profile of CTX-
enzyme. Colony-blot hybridization revealed that 53/200 M-positive isolates. Most CTX-M-positive isolates (39/53)
(26.5%) ESBL-positive strains carried a CTX-M-type gene. showed high MIC values (≥32 mg/l) for cefotaxime with bor-
The first CTX-M-positive strain was detected in December derline values (1–2 mg/l) for ceftazidime. The remaining 14
1999 from urinary sample of an outpatient and was found to isolates had also high MICs for ceftazidime, i.e., ≥4 mg/l.
carry the blaCTX-M-1 gene. Over the 5-year period, CTX-M- All CTX-M-positive isolates were susceptible to imipenem
positive isolates were obtained from both inpatients (n = 29) (MIC < 1 mg/l).
and outpatients (n = 24). As shown in Table 1, increasing The following resistance traits were found in associa-
numbers of CTX-M-positive strains were observed from tion with CTX-M expression: tetracycline in 41/53 isolates
1999 to 2003. Strains carrying the blaCTX-M-1 gene were (77.3%), co-trimoxazole in 33/53 (62.3%), ciprofloxacin in
detected throughout the entire 5-year period, whereas strains 26/53 (49.0%) and gentamicin in 10/53 (18.9%) isolates. One
carrying the blaCTX-M-15 gene only appeared in 2003. isolate was resistant to amikacin, all the other were suscepti-
Differences were seen in the double-disk synergy assays ble.
performed on CTX-M-1- and CTX-M-15-positive E. coli iso- Complete clinical records of 29 hospitalized patients were
lates (Fig. 1). In particular, compared with CTX-M-1-positive precisely investigated. Analysis of clinical data (Table 3)
strains, those positive for CTX-M-15 had a reduced inhibi- showed that most patients infected with CTX-M-positive
tion zone of ceftazidime (i.e., an increased hydrolytic activity strains were over 60 years of age and there was no gender

Fig. 1. Double-disk synergy test in E. coli isolates producing the CTX-M-1 (panel A) or CTX-M-15 (panel B) enzyme. Each disk contained 30 ␮g of either
cefotaxime, aztreonam, ceftazidime or ceftriaxone. Disks were placed 25 mm apart (centre to centre) around a disk containing amoxicillin (20 ␮g) plus clavulanic
acid (10 ␮g).
160 G. Brigante et al. / International Journal of Antimicrobial Agents 25 (2005) 157–162

Table 2
Susceptibility profile of CTX-M-1-positive and CTX-M-15-positive E. coli isolates
Beta-lactam Number of isolates per each MIC value (mg/l)
<1 1 2 4 8 16 32 64 ≥128
A. Susceptibility profile of CTX-M-1-positive E. coli isolates (n = 51)
Cefotaxime 18 22 11
Ceftazidime 6 33 8 4
Ceftriaxone 8 30 13
Cefepime 5 14 21 9 2
Aztreonam 2 8 14 11 16
Piperacillin 51
Ampicillin/sulbactam 1 12 38
Amoxicillin/clavulanate 1 1 24 18 7
Piperacillin/tazobactam 29 15 3 4
Imipenem 51
B. Susceptibility profile of CTX-M-15-positive E. coli isolates (n = 2)
Cefotaxime 2
Ceftazidime 1 1
Ceftriaxone 2
Cefepime 2
Aztreonam 2
Piperacillin 2
Ampicillin/sulbactam 2
Amoxicillin/clavulanate 2
Piperacillin/tazobactam 2
Imipenem 2

preference. Overall, isolates were obtained from the urinary Kluyvera spp. [20] and have only limited structural homol-
tract (42/53; 79.2%), wounds (n = 7), lower respiratory tract ogy with classical TEM- and SHV-derived ESBL [21]. A few
(n = 2) and blood (n = 2). CTX-M-positive E. coli isolates data are available on the prevalence of CTX-M-type genes
were obtained from patients hospitalized in several different among clinical isolates of gram-negative rods. Recent reports
wards, suggesting that there was no clonal relation between from Spain [22], France [10,23], Russia [9], Canada [24] and
the infecting strains. Clonal relations among different strains Taiwan [25] indicate the prevalence of CTX-M-type genes
were not detected by molecular typing with the ERIC2 PCR among ESBL-positive clinical isolates of E. coli as ranging
method [16]. Previous hospitalization during the 12-month from 1 to 81%. CTX-M-positive enterobacterial isolates have
period, preceding the current illness, was documented been reported sporadically from Italy [12,13].
in 33/53 patients. Eighteen of 53 patients had received This report provides data on the prevalence and charac-
cephalosporins within 2 month of the current infection. terization of CTX-M-type enzymes among clinical E. coli
Notably, the first patient infected with a CTX-M-15-positive isolates obtained over a 5-year period in Northern Italy. Data
strain had been given ceftazidime within 2 months of show that the isolation of CTX-M-positive E. coli increased
diagnosis. Hospitalized patients had a wide spectrum of from 1 in 1999 to 26 in 2003. In 2003, CTX-M-type genes
different diseases with the Charlson weighted index ranging accounted for 38.2% of ESBL-positive E. coli isolates. DNA
from 1 to 6. Considering both inpatients and outpatients, sequencing revealed that most isolates (51/53) carried the
the same bacterial pathogen was isolated on two or more blaCTX-M-1 gene. For the first time in Italy, the blaCTX-M-15
subsequent occasions in 15/53 (28.3%) patients. Of these, gene was found in two E. coli isolates. It is of interest that
12/15 (80%) had recurrent urinary tract infection (UTI). the resistance phenotypes associated with blaCTX-M-1 and
For epidemiological analysis, repeated isolates have been blaCTX-M-15 genes could be suspected (both by diffusion and
considered as single cases. microdilution assays) based on the different ability of the
carrier strain to hydrolyze ceftazidime and enhanced activ-
ity to other cephalosporins. Variants such as CTX-M-15 are
4. Discussion thought to evolve for their improved activity against cef-
tazidime [3] and a French report indicates that CTX-M-15
Since 1983 when ESBL-mediated resistance was first enzymes are becoming increasingly prevalent in K. pneumo-
detected in Germany [18], infections caused by ESBL- niae and E. coli [10].
producing enterobacteria have been reported world-wide es- At our Institution, CTX-M-positive strains were isolated
pecially in Klebsiella pneumoniae and E. coli [19]. CTX- from both inpatients (n = 29) and outpatients (n = 24). Though
M-type enzymes may be derived from a progenitor gene of the latter finding might indicate the spread of CTX-M-type
 G. Brigante et al. / International Journal of Antimicrobial Agents 25 (2005) 157–162 161

Table 3
Clinical data of 29 hospitalized patients with infections caused by CTX-M-positive E. coli isolates
Isolate Age Sex Year Clinical Admission Previous Previous use of Diagnosis at Charlson 2 or more
sample ward hospitalizationsa cephalosporinsb admission weighted isolates
index
0014/00 85 F 2000 Sputum Nephrology Yes CRO LRTI in CRF 2 No
0158/00 85 F 2000 Urine ICU Yes No Rectal cancer 3 No
0429/00 72 M 2000 Urine Urology Yes No Bladder cancer 6 No
0638/00 78 F 2000 Urine Cardiosurgery Yes CRO Aortic valvulopathy 1 No
0649/00 47 F 2000 Urine Gynaecology Yes No Endometrial cancer 2 Yes
0068/01 52 F 2001 Urine Gynaecology Yes No Endometrial cancer 2 No
0909/01 75 F 2001 Urine Nephrology Yes CAZ CRF 4 No
1115/01 70 M 2001 Wound ICU Yes No COPD 1 Yes
1154/01 50 M 2001 BAL ICU Yes CRO Encephalorrhagia 1 Yes
2346/01 52 M 2001 Urine Nephrology Yes CRO CRF 2 No
2525/01 84 M 2001 Wound ICU No No CRF 2 No
0064/02 81 F 2002 Urine Geriatrics Yes CRO Rectorrhagia 1 No
0287/02 5 M 2002 Urine Paediatries No No UTI ND No
0343/02 64 M 2002 Blood Infectivology Yes No Spondylodiscitis 1 Yes
0373/02 65 M 2002 Blood Medicine Yes No Amyloidosis 1 No
0547/02 56 F 2002 Wound Surgery Yes CAZ Colonic cancer 5 No
0687/02 82 F 2002 Urine Geriatrics No No Heart failure 2 No
0070/03 76 F 2003 Wound Radiotherapy Yes CRO Squamous cell car- 6 No
cinoma
0458/03 64 M 2003 Urine Infectivology Yes No Tuberculosis 4 Yes
5556/03 70 F 2003 Wound Medicine Yes No Septic ulcer 5 No
0687/03 54 M 2003 Wound Surgery Yes No Colonic cancer 5 No
0689/03 75 F 2003 Urine Nephrology Yes FEP Acute renal failure 5 No
0747/03 1 M 2003 Urine Paediatries Yes No UTI ND Yes
0843/03 90 F 2003 Urine Geriatrics Yes CRO, CAZ Encephalorrhagia 5 No
0927/03 63 M 2003 Wound Surgery Yes No Peptic ulcer 4 No
A10/03 79 M 2003 Urine Cardiosurgery No CEF Coronary artery by- 2 No
pass
3130/03 62 M 2003 Urine Urology Yes CRO Renal transplant 4 Yes
A70/03 62 M 2003 Urine Cardiosurgery Yes CEF Coronary artery by- 1 No
pass
7024/03 92 F 2003 Urine Orthopaedics No CEF Multiple fractures 1 No
Note: BAL, bronchoalveolar lavage; ICU, intensive care unit; CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; CEF, cefazolin; LRTI, lower respiratory
tract infection; COPD, chronic obstructive pulmonary disease; CRF, chronic renal failure UTI, urinary tract infection; ND, not determined.
a Preceding 12-months.
b preceding 2-months.

determinants in the community, clinical data revealed that should be discouraged in patients with underlying diseases
most patients (including the group of outpatients) were over and who have been hospitalized during the preceding year.
60-year-old, had been hospitalized more than once in differ- CTX-M-positive isolates were consistently susceptible to
ent hospital wards and some of them (18/53) had received imipenem, thus, carbapenems appear to represent the most
cephalosporins within 2 months of diagnosis. In particular, effective therapeutic approach. Since 52/53 isolates were sus-
the first patient infected by a CTX-M-15-positive strain had ceptible to amikacin and 47/53 to piperacillin-tazobactam
received ceftazidime. Taken together these data suggest that (MIC ≥ 4 mg/l), both drugs could be valuable therapeutic al-
CTX-M-positive E. coli isolates originated in the hospital en- ternatives.
vironment. Since drug-resistant clonal strains of E. coli have sThe results suggest that diffusion of CTX-M genes
been shown to cause UTI in the USA, the possible clonal ori- among enterobacteria may be associated with the spread
gin of CTX-M-positive isolates was investigated [26]. Typing of mobile elements carrying other resistance determinants.
by the ERIC2 PCR method tended to exclude clonal relations Plasmid-mediated transfer of the CTX-M-15 determinant
between the investigated isolates, suggesting that the diffu- in enterobacteria has been reported from Taiwan [27],
sion of blaCTX-M could be related to mobile genetic elements. while phage-mediated transfer of ESBL genes could not be
The majority of multidrug resistant strains caused re- detected in the environment [28]. The CTX-M-15 enzyme
current infection, especially UTI. In particular, 62.3% of belongs to the CTX-M-1 cluster and derives from CTX-M-3
strains were resistant to co-trimoxazole, 49.0% were re- by a single Asp-240-Gly substitution [29]. Thus, selective
sistant to ciprofloxacin. Thus, use of these two oral drugs pressure from the extensive use of oxyimino cephalosporins
162 G. Brigante et al. / International Journal of Antimicrobial Agents 25 (2005) 157–162

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