Prevalence and Mechanisms of Broad-Spectrum β-Lactam Resistance in Enterobacteriaceae: a Children's Hospital Experience

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2008, p. 3909–3914
0066-4804/08/$08.00?0doi:10.1128/AAC.00622-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 11
Prevalence and Mechanisms of Broad-Spectrum ?-Lactam Resistance
in Enterobacteriaceae: a Children’s Hospital Experience?
Xuan Qin,1,3* Danielle M. Zerr,2,3Scott J. Weissman,2,3Janet A. Englund,2,3Donna M. Denno,2,3
Eileen J. Klein,2,3Phillip I. Tarr,4Justin Kwong,1Jennifer R. Stapp,1
Luis G. Tulloch,3and Emmanouil Galanakis1
Microbiology Laboratory, Department of Laboratory Medicine, Children’s Hospital and Regional Medical Center, Seattle, Washington1;
Department of Pediatrics, University of Washington,2and University of Washington School of Medicine,3Seattle, Washington; and
Departments of Pediatrics and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri4
Received 12 May 2008/Returned for modification 8 July 2008/Accepted 22 August 2008
The objective of this study was to investigate the trends and patterns of resistance in ?-lactamase-producing
members of the family Enterobacteriaceae in a children’s hospital over a 9-year period (1999 to 2007). Clinically
significant isolates of the Enterobacteriaceae were screened for patterns of broad-spectrum resistance to
?-lactams. The strains likely to be resistant were subsequently confirmed by an inhibitor-based disc test. The
plasmid-mediated resistance determinants in these isolates were identified by PCR and by in vitro transfor-
mation, which successfully reproduced the AmpC phenotype unrestricted by the species of the host organisms.
Among 8,048 Enterobacteriaceae isolates belonging to the four chromosomal ampC-negative or -nonfunctional
genera, 86 (1.07%) isolates (56 Escherichia coli isolates, 22 Klebsiella species isolates, 1 Proteus mirabilis isolate,
and 7 Salmonella species isolates) exhibited broad-spectrum ?-lactam resistance patterns. These organisms
collectively produced three classes of ?-lactamases, including class A extended-spectrum ?-lactamases (n ?
47), class C or AmpC ?-lactamases (n ? 36, including 4 isolates that produced both class A and class C
enzymes), and class A or B carbapenem-hydrolyzing ?-lactamases (n ? 3). The proportion increased from
0.46% during the first 3 years to 1.84% during the last 3 years (relative risk [RR], 4.04; 95% confidence interval
[CI], 2.28 to 7.42; P < 0.001). The increase was mainly due to the emergence of a plasmid-mediated blaCMY-2
?-lactamase, the incidence of which increased from 0.11% during the first 3 years to 0.96% during the last 3
years (RR, 9.11; 95% CI, 2.76 to 30.1; P ? 0.001). Class A-type resistance increased slightly during the study
period, from 0.35% during the first 3 years to 0.85% during the last 3 years (RR, 2.42; 95% CI, 1.15 to 5.07; P ?
0.02). A Proteus mirabilis strain was documented to possess a novel blaDHAdeterminant. Of special concern,
three carbapenemase-producing isolates were identified between 2003 and 2006. The infections caused by
resistant isolates of the Enterobacteriaceae mainly affected hospitalized patients with underlying conditions;
however, 19 (22%) episodes were of community onset in otherwise well children. The rate of resistance to
broad-spectrum ?-lactams among isolates of the Enterobacteriaceae is increasing in children in both hospital-
and community-acquired settings, and the resistance is driven largely by plasmid-mediated AmpC ?-lacta-
mases. These data have important implications for empirical antimicrobial strategies targeting serious pedi-
atric infections. Further study of this problem is warranted.
The ever increasing variety of ?-lactamases produced by iso-
lates of the family Enterobacteriaceae raises concerns about our
dependenceon?-lactamdrugsandtheemergenceofpanresistant
species. Rapidly emerging ?-lactamases include extended-spec-
trum ?-lactamases (molecular class A ?-lactamases and, to a
lesser extent, class D ?-lactamases in the Enterobacteriaceae),
AmpC ?-lactamases (class C), and carbapenem-hydrolyzing
?-lactamases (mainly those of classes B, A, and D) (2, 4, 12, 18,
20). Escherichia coli and Klebsiella spp. producing class A extended-
spectrum ?-lactamase (ESBL) enzymes have been well docu-
mented in the United States, but the emergence of blaCTX-M
determinants is a recent finding (2, 16). Plasmid-encoded blaCMY,
blaDHA, and blaACCdeterminants producing class C enzymes are
being described with increasing frequencies.
Class A ESBLs and class C (AmpC) ?-lactamases hydrolyze
extended-spectrum cephalosporins, but AmpC ?-lactamases
are able to actively hydrolyze cephamycins and are resistant to
inhibition by clavulanate or other ?-lactamase inhibitors in
vitro (4, 18, 20). Carbapenemases have a broader range activity
than class A or class C ?-lactamases and affect the activities of
carbapenems as well as those of cephalosporins (4, 13, 18, 20).
The spread of most broad-spectrum ?-lactamases is facilitated
by transferable and transconjugable plasmids, which often
carry other resistance genes by means of their integron archi-
tecture (12). Genes encoding ESBLs and carbapenemases are
located on plasmids, while historically, genes encoding AmpC
?-lactamases have primarily been located on the chromosomes
of certain genera of the family Enterobacteriaceae (17). Re-
cently, however, ampC genes were documented to episomalize
into plasmids and disseminate into various species of the family
Enterobacteriaceae (29). The plasmid-borne ampC gene is of-
ten constitutively expressed and may confer high-level resis-
tance to ?-lactams (20).
Resistant isolates of the Enterobacteriaceae have recently
* Corresponding author. Mailing address: Microbiology Laboratory,
Department of Laboratory Medicine, 6P-1, Children’s Hospital and
Regional Medical Center, 4800 Sand Point Way, NE, Seattle, WA
98105-0371. Phone: (206) 987-2586. Fax: (206) 987-3840. E-mail: xuan
.qin@seattlechildrens.org.
?Published ahead of print on 2 September 2008.
3909

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emerged as a problem in adults, both in hospital settings and in
community settings. The Enterobacteriaceae are also major
pathogens in neonates, infants, and children, although little is
known about the broad-spectrum ?-lactamase-producing
strains in this specific age group (7, 9, 19, 25–26, 28). The
spread of ?-lactam-resistant Enterobacteriaceae in children is
of particular importance, since fluoroquinolones are not ap-
proved for use in this age group and are not considered first-
line agents for use in this age group. Given the clinical impact
of Enterobacteriaceae during childhood, we set out to investi-
gate the epidemiology of highly resistant organisms and the
molecular determinants of resistance in a pediatric hospital
setting.
MATERIALS AND METHODS
Setting. Children’s Hospital and Regional Medical Center (CHRMC) in
Seattle, WA, is a 250-bed tertiary-care pediatric hospital with 13,000 patient
admissions annually. The study included specimens collected over a 9-year pe-
riod (from January 1999 to December 2007). The study was approved by the
CHRMC Institutional Review Board.
Isolation and antibiotic susceptibility testing. In total, 8,048 isolates belonging
to only five species, E. coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus
mirabilis, and Salmonella enterica, were included. These isolates met the com-
monly used criteria (10) for clinically significant isolates (an organism isolated
from a sterile body site or a significant quantity of growth from nonsterile sites,
such as urine, wounds, or tracheal aspirates) and received a full microbiological
evaluation, including the performance of antibiotic susceptibility tests. Organ-
isms isolated from the same patient at intervals of ?7 days were included. The
majority of specimens were obtained to evaluate specific illnesses. In 2006,
surveillance for multiresistant E. coli and/or Klebsiella spp. from patients hospi-
talized with cancer or for bone marrow or solid organ transplantation was
initiated. In these cases, a stool or rectal swab sample for culture was obtained
upon admission and periodically (?1 per month) after extended hospitalization.
Of the 8,048 total isolates, 86 were further characterized for their broad-
spectrum ?-lactam resistance phenotypes. Clinical samples were processed in the
CHRMC Microbiology Laboratory according to standard operating procedures.
Antibiotic susceptibility was determined by the standard disc diffusion method on
Mueller-Hinton agar or by determination of the MIC by use of the Vitek system
(11, 13, 24). The zones of inhibition were interpreted according to the guidelines
of the Clinical and Laboratory Standards Institute (CLSI; formerly the NCCLS)
(3). The susceptibilities of all isolates to ampicillin, amoxicillin-clavulanic acid,
aztreonam, cefazolin, cefuroxime, cefotetan (determined only with the Vitek
system), ceftazidime, ceftriaxone, cefepime, meropenem, piperacillin-tazobac-
tam, ciprofloxacin, gentamicin, and sulfamethoxazole-trimethoprim were deter-
mined; susceptibility to nitrofurantoin was tested only for the urinary isolates.
Characterization of ?-lactamase-producing strains. On the basis of the crite-
ria of the CLSI, the paired disc diffusion method was applied on Mueller-Hinton
agar by using multiple discs with ceftazidime (30 ?g), cefotaxime (30 ?g), ceftaz-
idime-clavulanic acid (30/10 ?g), cefotaxime-clavulanic acid (30/10 ?g), and
cefpodoxime (30 ?g); and tests with cefepime (30 ?g), imipenem (10 ?g), and/or
meropenem (10 ?g) were often repeated to rule out the presence of potential
carbapenemase producers. ESBL production was defined as an increase in the
zone of inhibition of ?5 mm with either ceftazidime or cefotaxime discs when
they were tested in combination with discs containing clavulanic acid (3, 11). The
class C ?-lactamase resistance or AmpC phenotype was defined as a change in
the diameter of the zone of inhibition of ?5 mm between discs containing the
aforementioned drugs in combination with discs containing clavulanate, in ad-
dition to a likely susceptible zone of inhibition around the cefepime disc. Isolates
E. coli ATCC 25922 and K. pneumonia ATCC 700603 were used as control
strains.
PCR for ?-lactamase genes. For those isolates that met the criteria for either
the ESBL resistance or the AmpC phenotype, a five-gene panel for the ampli-
fication of the blaCMY, blaDHA, blaACC, blaTEM, and blaCTX-Mgenes was applied.
The primers for blaIMPand blaKPCwere used only with those isolates that
exhibited resistance to carbapenems. The specific PCR primers and the previ-
ously described assay methods used and are listed in Table 1. A subset of
amplicons showing the expected molecular mass was further sequenced, and a
search for the homology of the sequences with the sequences of specific genetic
determinants was performed.
Plasmid content and transformation. Plasmid DNA was extracted from the
isolates of E. coli, K. pneumoniae, K. oxytoca, and Salmonella spp. that tested
positive for blaCMYby use of a QIAprep Spin Miniprep kit (Qiagen, Hilden,
Germany). The E. coli competent cells (Qiagen EZ kit) were transformed, and
the transformants were selected on Luria-Bertani agar containing ceftazidime
(10 ?g/ml) (GlaxoSmithKline, Lisbon, Portugal) (9). The E. coli transformants
were reexamined both phenotypically for the reproducibility of the susceptibility
patterns and genotypically for the presence of the blaCMYdeterminants com-
pared to the phenotypes and genotypes of the corresponding host organisms.
Clinical variables. Clinical data were collected from the medical records of
children receiving care at CHRMC. The clinical variables included gender, age,
the duration of hospitalization, the number of previous hospitalizations, the
history of the patient’s antibiotic regimen prior to sampling, the presence of
catheters, underlying conditions, the site of sampling, the setting of the sampling,
and the outcome of infection.
Statistics. Statistical analysis was performed with the EpiInfo software pro-
gram (version 3.4; Centers for Disease Control and Prevention). The proportions
between groups were compared by the chi-square test. Relative risks (RRs; with
Taylor series 95% confidence intervals [CIs]) were calculated.
RESULTS
Prevalence of ?-lactamase-producing Enterobacteriaceae.
Eighty-six (1.07%) isolates of the five specified species from 79
patients met the criteria of the CLSI (according to either the
zone of inhibition or the MIC) for further drug susceptibility
testing to determine the mechanisms of ?-lactam resistance
(Table 2). The isolates were derived from multiple specimen
types (53 from urine specimens; 9 from blood specimens; 8
from stool specimens; 3 from wound or abscess specimens; 2
from peritoneal fluid specimens; and 1 each from cerebrospi-
nal fluid, eye swab, genital swab, tracheal aspirate, and gastro-
intestinal fluid specimens). In three cases, the same strain was
isolated from more than one site (only one strain from each of
these patients was used), while more than one species was
isolated from four urine specimens and a single blood culture
(all bacterial species from these patients were included). Two
earlier isolates had no recorded links to patients. The fre-
quency of resistant organisms increased significantly during the
9-year study period (P ? 0.001, chi-square test for trend). The
proportion increased from 0.46% during the first 3 years to
1.84% during the last 3 years (RR, 4.04; 95% CI, 2.28 to 7.42;
P ? 0.001). This increase was observed both for E. coli isolates,
the prevalence of which increased from 0.41% to 1.82% (RR,
4.67; 95% CI, 2.16 to 9.25; P ? 0.001), and for non-E. coli
TABLE 1. Specific primers used for molecular amplification and
partial sequencing of the bla genes
bla geneOligonucleotide sequence
Product
length
(bp)
Reference
or source
blaCMY-2
TGG CCA GAA CTG ACA GGC AAA
TTT CTC CTG AAC GTG GCT GGC
AAC TTT CAC AGG TGT GCT GGG T
CCG TAC GCA TAC TGG CTT TGC
AAC AGC CTC AGC AGC CGG TTA T
TTC GCC GCA ATC ATC CCT AGC
GAA AGG GCC TCG TGA TAC GC
TCA TCC ATA GTT GCC TGA CTC C
CGA TGT GCA GTA CCA GTA A
TTA GTG ACC AGA ATC AGC GG
GTA CAG TCT ATG CCT CG
GTA MGT TTC AAG AGT GAT GC
GCT ACA CCT AGC TCC ACC TTC
ACA GTG GTT GGT AAT CCA TGC
46121
blaDHA
40521
blaACC
34621
blaTEM
1,0051
blaCTX-M
5852
blaIMP
750This study
blaKPC
988This study
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isolates, the prevalence of which increased from 0.62% to
1.89% (RR, 3.06; 95% CI, 1.01 to 9.25; P ? 0.04).
Class A ESBL-type resistance. Overall, 47 (0.58%) isolates
were identified as ESBL producers, and 39 of these were avail-
able for molecular characterization. Genes encoding blaTEM
were detected in 26 isolates, and those encoding blaCTX-Mwere
detected in 13 isolates; 5 of these isolates contained both de-
terminants, and 5 isolates were negative for both determinants
(Table 2). The rate of class A-type resistance increased from
0.35% during the first 3 years to 0.85% during the last 3 years
(RR, 2.42; 95% CI, 1.15 to 5.07; P ? 0.02) (Fig. 1). The ESBL
producers often demonstrated coresistance to gentamicin
(45%), ciprofloxacin (46%), and sulfamethoxazole-trimetho-
prim (85%). Coresistance to all three agents occurred in 10%
of the ESBL producers. Of the class A ESBL producers, 40%
showed susceptibility to cefepime in vitro, although the clinical
utility of this finding remains to be determined.
Class C AmpC-type resistance. AmpC-type resistance was
observed in 36 (0.45%) isolates. The frequency of AmpC-type
resistance increased sharply over the study period, from 0.11%
during the first 3 years to 0.96% during the last 3 years (RR,
9.11; 95% CI, 2.76 to 30.1; P ? 0.001) (Fig. 1). Among these
isolates, 29 were positive for blaCMY, 6 were positive for
blaDHA, and 1 remaining E. coli isolate was negative for all
three ampC determinants (blaCMY,blaDHA, and blaACC) (Ta-
ble 2). Two E. coli isolates and one K. pneumoniae isolate
contained dual resistance determinants (blaCMYand blaTEM),
and a single K. pneumoniae isolate contained triple resistance
determinants (blaDHA, blaTEM, and blaCTX-M). Sequencing of
the blaCMYamplification products revealed blaCMY-2-like cod-
TABLE 2. Characterization of gene determinants in 86 isolates of the Enterobacteriaceae exhibiting resistance to
broad-spectrum ?-lactamsa
Isolate and resistance
determinant
All isolates AmpC-producing strains ESBL-producing strains
Carbapenemase-producing
strains
No. of strains that
met the CLSI
criteria/total no.
tested (%)
No. of
strains
isolated
from
urine
No. of strains that
met the CLSI
criteria/total no.
tested (%)
No. of
strains
isolated
from
urine
No. of strains that
met the CLSI
criteria/total no.
tested (%)
No. of
strains
isolated
from
urine
No. of strains that
met the CLSI
criteria/total no.
tested (%)
No. of
strains
isolated
from
urine
Escherichia coli
blaCMY
blaDHA
blaTEM
blaCTX-M
blaIMP
Negative for all
determinants
Missing isolates
56/6,097 (0.9)4422/56 (39.2)
19
2
2
16
14
1
2
32/56 (57.1) 282/56 (3.6)0
21
10
20
10
2
1144
1
Klebsiella pneumoniae
blaCMY
blaDHA
blaTEM
blaCTX-M
blaKPC
Missing isolates
17/941 (1.8)116 (35.3)
3
3
2
1
4
2
2
2
1
10 (58.8)71 (5.9)0
3
2
2
21
53
Klebsiella oxytoca
blaTEM
Negative for all
determinants
Missing isolates
5/398 (1.3)10 (0) 5 (100)
2
1
1
1
0 (0)
2
Proteus mirabilis
blaDHA
1/397 (0.25)1 1 (100)
1
1
1
0 (0)0 (0)
Salmonella spp.
blaCMY
7/215 (3.3)0 7/7 (100)
7
0 (0) 0 (0)
Total86/8,048 (1.1)57 36 (41.9) 2147 (54.6) 363 (3.5)0
aThe resistant isolates were from among 8,048 isolates of the Enterobacteriaceae isolated from clinical specimens at CHRMC from 1999 to 2007. More than one
determinant may be present in some isolates.
FIG. 1. Number of Enterobacteriaceae strains producing class A
ESBLs, class C AmpC, or carbapenem-hydrolyzing ?-lactamases. (?),
positive.
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ing sequences in all 29 blaCMY-positive isolates. Coresistance
to other classes of antibiotics, such as gentamicin (17%), cip-
rofloxacin (19%), and sulfamethoxazole-trimethoprim (39%),
appeared to be less pronounced in AmpC producers than in
ESBL producers.
Our first blaCMY-possessing isolate was a K. pneumoniae
strain isolated in 2003 from the urine of an 18-year-old female
patient with neurogenic bladder. Several months later, two
blaCMY-possessing Salmonella serovar 4,[5],12:i:? strains were
isolated from the stools of two children; these were related to
a food-borne outbreak in King County, WA, in the spring of
2004. Later in 2004, two E. coli strains were isolated from two
children with prolonged hospitalizations: one isolate from the
urine of a child with myelomeningocele and one isolate from a
wound of a child with neuroblastoma. Following this increase
in the incidence of blaCMY-possessing isolates, AmpC-type re-
sistance became more common than the previously dominant
class A ESBL-type resistance (Fig. 1).
Plasmids carrying blaCMYthat were extracted from E. coli
and Salmonella isolates were transferable in vitro. The AmpC
phenotype and its corresponding genetic determinants were
reproducible in E. coli transformants obtained with a Qiagen
EZ kit by susceptibility testing and PCR. The cotransfer of
antibiotic resistance to other antibiotics, such as gentamicin
and sulfamethoxazole-trimethoprim, was not observed in the
15 E. coli transformants positive for blaCMYobtained with the
Qiagen EZ kit.
Isolates carrying both class A and class C resistance deter-
minants. Four organisms (two K. pneumoniae isolates and two
E. coli isolates) contained both class A and class C determi-
nants. These organisms, which were isolated from cultures of
urine from unrelated patients, exhibited multidrug resistance
phenotypes. Two patients had histories of recurrent urinary
tract infections (UTIs), and the resistant isolates (one E. coli
isolate and one K. pneumoniae isolate, both of which carried
blaCMYand blaTEM) were each associated with a single episode
of infection. The K. pneumoniae strain was sensitive only to
carbapenems and was isolated from a child who had under-
gone a urological procedure. An E. coli isolate that carried
blaCMYand blaTEMand that was also gentamicin resistant was
isolated from a third patient who had undergone bone marrow
transplantation. The last patient had multiple congenital
anomalies, and both an E. coli isolate (carrying blaTEMand
blaCTX-M) and a K. pneumoniae isolate (carrying blaDHA,
blaTEM, and blaCTX-M) were isolated from the patient. These
two isolates were susceptible to both carbapenems and cipro-
floxacin.
Resistance to carbapenems. Two E. coli and one K. pneu-
moniae strains were found to be resistant to all ?-lactams,
including carbapenems. The two E. coli strains were found to
contain the blaIMP-4gene, and the K. pneumoniae strain was
positive for blaKPC-2. The two E. coli strains did not appear to
be related. They were isolated 1 year apart (in December 2003
and December 2004, respectively), and the patients’ hospital
stays did not overlap. In addition, the patterns of susceptibility
of the two isolates to aminoglycosides and quinolones were
different. The K. pneumoniae strain containing blaKPC-2was
isolated in the fall of 2006 and was susceptible only to tigecy-
cline and colistin. Both of the children from whom IMP-4-
producing E. coli strains were isolated had previously been
seen in other institutions (one in Alaska and the other in
Mexico), and their antibiotic treatment histories were not
available. The child from whom the KPC-2-producing K. pneu-
moniae strain was isolated had arrived in Seattle shortly after
a prolonged hospitalization in the northeastern United States.
Patients. The infected patients ranged in age from 4 days to
25.5 years (median, 5.5 years). Among the infants aged ?1
year, resistant isolates were more common in males (male-to-
female ratio, 1.8:1), but beyond infancy, females were overrep-
resented (female-to-male ratio, 2.2:1; P ? 0.05). The majority
of the patients had chronic underlying conditions (60/79, or
76%), including urological, neurological, and hematological
diseases requiring multiple hospitalizations, indwelling cathe-
ters, and multiple antibiotic regimens. Seventeen patients had
severe urogenital diseases, 16 had leukemia, 4 had solid tu-
mors, 5 had myelomeningocele, and 5 had undergone trans-
plantation. Of the 79 cases, UTIs appeared to be the most
common clinical manifestation (53 isolates were obtained from
urine specimens). Urine was the most common source of E.
coli (77%) and K. pneumoniae (65%) isolates (Table 2). The
proportion of E. coli isolates obtained from urine was high
regardless of the determinant of resistance (14/19 isolates
[73%] from urine for blaCMYversus 20/21 isolates [95%] from
urine for blaTEMand 10/10 isolates [100%] from urine for
blaCTX-M; Table 2). A similar pattern was observed for the K.
pneumoniae isolates. Only one of the five K. oxytoca strains was
isolated from urine. All seven Salmonella isolates were associ-
ated with gastroenteritis, and three of these isolates were
linked with food-borne outbreaks. The child from whom the
KPC-2-producing K. pneumoniae strain (a blood isolate) was
obtained was the only patient who appeared to have died as a
direct result of the infection.
Resistance in the community setting. Community-acquired
infections were suggested in 19 (22.1%) cases on the basis of
the patients’ clinical histories. Seven of these children were
otherwise well and presented with nontyphoidal Salmonella
gastroenteritis over a period of 3 years. The Salmonella sero-
types isolated were S. enterica serotypes Heidelberg, Minne-
sota, Newport, and Typhimurium and Salmonella serovar
4,[5],12:i:?; and all isolates contained plasmid-encoded
blaCMY-2. Salmonella serotype Heidelberg was isolated from
both the stool and the blood of a single child. The remaining 12
children infected with resistant isolates of the Enterobacteria-
ceae presented with community-acquired UTIs which were
caused by E. coli in 10 cases (blaTEM, n ? 3; blaCMY, n ? 3;
blaCTX, blaDHA, and blaTEM-blaCTX, n ? 1 each) and by K.
pneumoniae in 2 cases (blaTEM, n ? 1; blaDHA-blaTEM-blaCTX,
n ? 1). Only four children in this category had a clear history
of antibiotic treatment.
Persistent colonization and recurrent infection. Persistent
colonization was found in five hematology-oncology patients
and one patient with short bowel syndrome. The longest period
of persistent colonization and recurrent infection (of both
urine and blood) documented to date was in the patient with
short bowel syndrome, who was colonized by an E. coli isolate
containing blaTEMfor more than 4 years. No single case of
clearance was documented by surveillance cultures. These six
cases with persistent colonization included all three patients
who were infected by the carbapenemase producers. Two of
the six patients were also simultaneously colonized by vanco-
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mycin-resistant enterococci. Overall, recurrent infections were
documented in at least 14 patients, 10 of whom had recurrent
UTIs, 3 of whom had chronic wound infections, and 1 of whom
had recurrent bloodstream infections. Two different isolates
were recovered from one patient. Among the 14 patients with
recurrent infections, 6 were hematology-oncology patients and
the remaining 8 had renal insufficiency or other chronic med-
ical conditions. Among the 15 organisms causing recurrent
infections, 8 produced class A ?-lactamases (blaTEM, n ? 4;
blaCTX, n ? 2; blaTEM-blaCTX, n ? 2) and 5 produced class C
?-lactamases (blaCMY, n ? 3; blaDHA, n ? 1; blaDHA-blaTEM-
blaCTX, n ? 1); no genetic determinant was found in 2 isolates.
DISCUSSION
We found a statistically significant increase in the rate of
resistance among isolates of the family Enterobacteriaceae over
a 9-year study period. The resistance patterns were character-
ized by a modest increase in the prevalence of ESBL-produc-
ing strains; by the sporadic, although worrisome, occurrence of
carbapenemase-producing strains; and by the sharp increase in
the incidence of plasmid-mediated AmpC-producing strains,
driven by the emergence of a plasmid containing blaCMY-2.
This is the first surveillance study of resistance among isolates
of the Enterobacteriaceae in the pediatric population of which
we are aware. Our findings suggest that plasmid-mediated
?-lactamase-producing isolates of the family Enterobacteria-
ceae are of increasing concern in pediatrics, both in the hos-
pital setting and in the community setting.
In our study, class A ESBL-producing Enterobacteriaceae
mainly possessed the blaTEMand the blaCTX-Mgenes. Entero-
bacteriaceae harboring transferable blaTEM, blaCTX-M, blaSHV
and blaOXAgenes have been reported in clinical isolates world-
wide (23, 25–26, 28, 30) and in commensal isolates in healthy
children in Bolivia and Peru (19). Among the various ESBL
enzymes, those of the CTX-M type have become predominant
worldwide in both hospital and community settings (2, 4).
Plasmid-mediated AmpC resistance has recently been re-
ported in clinical isolates of the Enterobacteriaceae worldwide
(6, 7, 9, 22, 23, 30–32). Few population-based studies have
reported on the frequency or clinical impact of these isolates,
and very little is known about the associated epidemiology in
pediatrics. In our study, the incidence of AmpC ?-lactamase
producers increased sharply due to the emergence of a blaCMY-2-
containing plasmid, and they appeared to be present in a wider
range of Enterobacteriaceae isolates than the class A ?-lacta-
mases were.
In our study, the most frequent determinants of resistance in
E. coli isolates were blaCMY-2(n ? 19), blaTEM(n ? 21), and
blaCTX-M(n ? 10). Although we did not detect epidemiolog-
ical links between the patients, the homogeneity suggests the
potential transmission of bacterial strains between patients.
The application of techniques for the determination of strain
relatedness may be warranted in future investigations in order
to reveal potential routes of transmission.
This study produced several novel or unusual findings, in-
cluding the isolation of a P. mirabilis strain containing a blaDHA
determinant from a pediatric patient; the finding of three car-
bapenemase-producing isolates of Enterobacteriaceae, espe-
cially IMP-4-producing E. coli isolates, in pediatric patients;
and the isolation of organisms with multiple determinants of
broad-spectrum resistance from a pediatric population. The
emergence of carbapenemase-producing Enterobacteriaceae,
especially blaKPCcarriers, has already been reported in New
York City and was subsequently reported in Europe and on
other continents (5, 12, 20); but few cases have been reported
in pediatric patients. Similarly, reports of organisms with mul-
tiple determinants of resistance are rare. In our study, six E.
coli strains each contained two class A resistance determinants
(blaTEMand blaCTX-M), while two E. coli strains and two K.
pneumoniae strains each contained both class A (blaTEMor
blaCTX-M) and class C (blaCMYor blaDHA) resistance determi-
nants. K. pneumoniae strains harboring genes for both class A
(blaSHV) and class C (blaDHA) resistance determinants were
recently reported in adults in Belgium (27). We are unaware of
reports of such organisms in pediatric patients. Genes encod-
ing ?-lactamases are frequently transferred on large plasmids
together with genes encoding resistance to aminoglycosides,
sulfonamides, doxycycline, and chloramphenicol (26). Multi-
drug resistance in the Enterobacteriaceae has crucial clinical
implications in pediatrics, as it renders the empirical regimens
(ampicillin plus gentamicin or ceftriaxone/cefotaxime) for the
treatment of septicemia in children ineffective. Multidrug re-
sistance trends require close monitoring, and the epidemiology
of such resistance requires a better understanding so that em-
pirical antimicrobial approaches may be effective, especially in
the setting of critical illness.
The persistent nature of colonization with broad-spectrum
?-lactam-resistant organisms was demonstrated in six patients
in our study who were monitored periodically by the use of
surveillance cultures. This has previously been demonstrated
by others (14). We now routinely perform rectal swab surveil-
lance cultures for patients undergoing bone marrow and solid
organ transplantation to rule out the presence of vancomycin-
resistant enterococci and broad-spectrum ?-lactam-resistant
Enterobacteriaceae. These data are then available to inform the
empirical management of invasive infections in these patients.
Resistant isolates of the Enterobacteriaceae have historically
been of nosocomial origin, and several factors have been
shown to contribute to their emergence (4, 8, 9, 11–15, 28).
However, community-acquired infections are increasingly rec-
ognized (2, 4, 14, 15, 23) and were quite common in our study,
in which eight children with UTIs caused by resistant Entero-
bacteriaceae had no record of a previous hospital stay and/or a
prolonged antibiotic treatment history. Further research on
multidrug-resistant Enterobacteriaceae isolates from the com-
munity setting is warranted.
Our findings demonstrate the growing problem of broad-
spectrum plasmid-mediated ?-lactam resistance in pediatric
Enterobacteriaceae isolates. The emergence of class C plasmid-
mediated blaCMYgenes among diverse genera of the Entero-
bacteriaceae and the isolation of carbapenemase-producing or-
ganisms from children are of concern. The emergence of such
a variety of resistant organisms poses a significant threat to
pediatric patients, for whom ?-lactams are the mainstay of
empirical therapy for critical illness and quinolones are not
commonly used as first-line therapies. We believe that the
judicious use of antimicrobials and the implementation of in-
fection control principles are important for the prevention of
the further spread of resistance in Enterobacteriaceae both in
VOL. 52, 2008 MULTIRESISTANCE IN PEDIATRIC PATIENTS3913

Page 6

the hospital and in the community. It is likely that such inter-
ventions are possible and will be most effective only when the
overall resistance rate is still low. The broad-spectrum plasmid-
mediated ?-lactam resistance trends in pediatric Enterobacte-
riaceae isolates require continued close monitoring, and fur-
ther study of the epidemiology of such resistance appears to be
warranted so that potential interventions that may be used to
halt the spread of resistance may be developed.
ACKNOWLEDGMENTS
We acknowledge the technical assistance of Lynn Stapp, Patrick
Abe, Scott Anderson, Joan Guzzo, and Treva Tsosie.
The research was conducted in part with a Marie Meyer Scholarship
in Clinical Microbiology awarded to Emmanouil Galanakis, who is a
pediatric infectious disease physician from the University of Crete,
Greece.
None of the authors has a potential conflict of interest.
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3914QIN ET AL.ANTIMICROB. AGENTS CHEMOTHER.