0095-1137/06/$08.00
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0
doi:10.1128/JCM.01916-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Clonality and Antimicrobial Resistance Gene Profiles of
Multidrug-Resistant
Salmonella enterica
Serovar Infantis Isolates from
Four Public Hospitals in Rio de Janeiro, Brazil
E. L. Fonseca,
1O. L. Mykytczuk,
4,5M. D. Asensi,
1E. M. F. Reis,
1L. R. Ferraz,
2F. L. Paula,
3L. K. Ng,
4,5and D. P. Rodrigues
1*
Bacteriology Department, Oswaldo Cruz Institute – FIOCRUZ, Rio de Janeiro, Brazil
1; Public Health Laboratory, Brasilia, Brazil
2;
Evandro Chagas Institute, Para, Brazil
3; National Microbiology Laboratory, Public Health Agency of Canada,
Winnipeg, Manitoba, Canada
4; and Department of Medical Microbiology, Faculty of Medicine,
University of Manitoba, Winnipeg, Manitoba, Canada
5Received 13 September 2005/Returned for modification 7 November 2005/Accepted 8 May 2006
In Brazil,
Salmonella enterica
serovar Infantis resistant to various antimicrobials, including cephalosporins,
has been identified as an etiological agent of severe gastroenteritis in hospitalized children since 1994. In this
study, 35 serovar Infantis strains, isolated from children admitted to four different Rio de Janeiro, Brazil,
hospitals between 1996 and 2001, were characterized by pulsed-field gel electrophoresis (PFGE) and
antimi-crobial susceptibility testing in order to determine their genetic relatedness and antimiantimi-crobial resistance
profiles. Thirty-four serovar Infantis strains were resistant to at least two antibiotic classes, and all 35 strains
were susceptible to fluoroquinolones, cephamycin, and carbapenem. Extended-spectrum beta-lactamase
(ESBL) screening by double-disk diffusion indicated that 32 serovar Infantis strains (91.4%) produced
beta-lactamases that were inhibited by clavulanic acid. Antimicrobial resistance gene profiles were determined by
PCR for a subset of 11 multidrug-resistant serovar Infantis strains, and putative ESBLs were detected by
isoelectric focusing. Ten serovar Infantis strains carried
bla
TEM,
catI
,
ant(3
ⴖ
)Ia
and/or
ant(3
ⴖ
)Ib
,
sulI
and/or
sulII
, and
tet
(D) genes as well as an integron-associated
aac(6
ⴕ
)-Iq
cassette. Eight strains possessed at least four
different beta-lactamases with pI profiles that confirmed the presence of both ESBLs and non-ESBLs. Our
PFGE profiles indicated that 33 serovar Infantis strains isolated from Rio de Janeiro hospitals came from the
same genetic lineage.
For many years, ampicillin, sulfamethoxazole-trimethoprim,
and chloramphenicol were the drugs of choice for the
treat-ment of severe
Salmonella
infections, but increasing rates of
resistance to these agents have significantly reduced their
ef-ficacies (28, 35). Subsequently, third-generation
cephalospo-rins, due to their pharmacodynamic properties as well as low
resistance levels in
Salmonella
, are being used to treat invasive
salmonellosis (5, 11).
In 1994, Asensi and Hofer reported the presence in Rio de
Janeiro, Brazil, of
Salmonella enterica
serovar Infantis strains
that were resistant to a growing number of antimicrobial
agents (6). Two years later, a nosocomial outbreak in a
neo-natal unit of one hospital (designated HC) was reported by De
Moraes et al. (13). The authors detected multidrug-resistant
serovar Infantis phenotypes, including resistance to
broad-spectrum cephalosporins that was transferred by a plasmid of
148 kbp. An investigation carried out from 1998 to 1999
re-ported an infection due to extended-spectrum beta-lactamase
(ESBL)-producing serovar Infantis in the neonatal unit of a
public hospital (HC) in Rio de Janeiro, Brazil, indicating
in-adequate infection control practices and nursery overcrowding
(30). Since then, multidrug-resistant serovar Infantis has been
isolated in three other public health hospitals (designated HA,
HB, and HD) of Rio de Janeiro, Brazil. Two are pediatric
reference hospitals that often see children from the western
and northern regions of the city, where parts of the population
have lower socioeconomic and sanitary conditions. Some
chil-dren were human immunodeficiency virus positive, and most
suffered from recurring infections and had histories of
rehos-pitalization. Although HC is a university-affiliated hospital and
HD is a reference hospital for cancer, both provide medical
care for patients with debilitating diseases such as AIDS and
diabetes. In addition, these patients are subjected to prolonged
hospitalizations that are often accompanied by the empirical
use and sometimes overuse of antimicrobial drugs (ampicillin
and/or cephalosporins and/or aminoglycosides). This led us to
monitor the prevalence and antimicrobial susceptibility of
se-rovar Infantis in hospitals in Rio de Janeiro, Brazil. The aims
of this research were to (i) determine the antimicrobial
sus-ceptibility patterns, (ii) identify the main mechanisms involved
in antimicrobial resistance, (iii) ascertain the presence and
spread of integron-carried resistance genes, and finally, (iv)
assess the macro-restriction fragment length polymorphisms
between multidrug-resistant serovar Infantis strains from those
hospitals.
MATERIALS AND METHODS
Bacterial strains. Serovar Infantis strains were isolated according to the method of Costa and Hofer (12), and the antigenic characterization was based on the Kauffmann-White scheme described by Poppof (29a). This study included 35
* Corresponding author. Mailing address: Laborato
´rio de
Entero-bacte
´rias, Departamento de Bacteriologia, Oswaldo Cruz Institute –
FIOCRUZ, Avenida Brasil, 4365 – Pavilha
˜o Rocha Lima, 3° andar,
Manguinhos – Rio de Janeiro, Brasil 21040-361. Phone: 55 21 2598
4277. Fax: 55 21 2270 6565. E-mail: [email protected].
2767
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serovar Infantis strains isolated from the stools or blood of children under 7 months who were admitted to four public hospitals (HA, HB, HC, and HD) in Rio de Janeiro, Brazil, from 1996 to 2001. Only one isolate per patient was included in the study.
Antimicrobial susceptibility testing and extended-spectrum beta-lactamase assay.Disk diffusion tests were performed according to Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Stan-dards) (26) recommendations by using disks (Oxoid Limited, Hampshire, England) impregnated with ampicillin (AMP; 10g), aztreonam (ATM; 30g), cephalothin (CEF; 30g), cefotaxime (CTX; 30g), ceftriaxone (CRO; 30g), ceftazidime (CAZ; 30g), cefoxitin (FOX; 30g), cefuroxime (CXM; 30g), cefepime (FEP; 30g), ciprofloxacin (CIP; 5g), chloramphenicol (CHL; 30g), streptomycin (STR; 10g), kanamycin (KAN; 10g), gentamicin (GEN; 10g), imipenem (IPM; 10g), nalidixic acid (NAL; 30g), trimethoprim-sulfamethoxazole (SXT; 25g), and tetracycline (TET; 30g). For quality control of the culture media and antimi-crobial disks,Escherichia coliATCC 25922,E. coliATCC 35218,Pseudomonas aeruginosaATCC 27853,Enterococcus faecalisATCC 29212, andStaphylococcus aureusATCC 25923 were tested under the same conditions and antimicrobials as was suggested by the CLSI (26).
The method described by the CLSI for “otherEnterobacteriaceae” was used to perform double-disk diffusion for the screening of ESBL-producing strains. Dou-ble-disk diffusion was performed with cephalosporin and cephalosporin/clavu-lanic acid combination disks (Oxoid Limited, England).Klebsiella pneumoniae
ATCC 700603 (positive) andE. coliATCC 25922 (negative) were used as control strains. In addition, the production of ESBLs in 11 serovar Infantis strains was
confirmed at the National Microbiology Laboratory, Public Health Agency of Canada, by using the Mast Diagnostics ESBL detection kit (Merseyside, United Kingdom) according to the manufacturer’s instructions.
Preparation of crude protein extracts and IEF.The 11 ESBL-positive isolates were grown in 2 ml of Mueller-Hinton broth at 37°C overnight, and cells were harvested by centrifugation at 16,000⫻gfor 2 min. After discarding the super-natant, cells were resuspended in 250l of 1% glycine and 30% glycerol and were sonicated twice for 30 s, with cooling of the cells on ice between sonications. Cell lysates were centrifuged at 16,000⫻g for 15 min. Supernatants were collected into clean tubes and stored at⫺20°C. Prior to isoelectric focusing (IEF), cell extracts were tested for beta-lactamase activity by adding 50l of 50
[image:2.585.47.541.80.455.2]g/ml nitrocefin stock solution (Oxoid Limited, England) to 17l of extract and then recording the time required for the reaction to turn dark pink. The optimal reaction time was 30 to 120 s. For reaction times of 5 s or less, the extract was diluted with phosphate buffer and retested. For isolates with reaction times of 5 min or more, another extract was prepared from a culture of greater density and the test was redone. For IEF, precast polyacrylamide IEF minigels (pH 3 to 10) (Bio-Rad Laboratories, Hercules, CA) were assembled in a vertical Bio-Rad Mini-Protean II electrophoresis unit. Cathode buffer (20 mM lysine-20 mM arginine) (Bio-Rad) was added to the middle chamber, the wells were flushed, and then 10l of crude extract was loaded in every second well. An IEF standard with pIs ranging from 4.45 to 9.6 (Bio-Rad) was used, and a marker composed of beta-lactamases of known isoelectric points (pIs) (blaTEM-1[pI 5.4],blaTEM-4 [pI 5.9],blaTEM-3[pI 6.3],blaSHV-3[pI 7.0], andblaSHV-2[pI 7.6]) was also used. Approximately 200 ml of anode buffer (7 mM phosphoric acid) (Bio-Rad) was
TABLE 1. PCR primers used to identify antimicrobial resistance genes and integrons in serovar Infantis
Gene or
integron Primer sequence 5⬘to 3⬘
a Reference strainb(plasmid) Source (reference)
tetA
F, GCT ACA TCC TGC TTG CCT TC; R, CAT AGA
TCG CCG TGA AGA GG
E. coli
D20-15 (pSL18)
S. Levy (21)
tetB
F, TTG GTT AGG GGC AAG TTT TG; R, GTA ATG
GGC CAA TAA CAC CG
E. coli
D20-16 (pRT11)
S. Levy (20)
tetC
F, CTT GAG AGC CTT CAA CCC AG; R, ATG GTC
GTC ATC TAC CTG CC
E. coli
D20-6 (pBR322)
S. Levy (20)
tetD
F, AAA CCA TTA CGG CAT TCT GC; R, GAC CGG
ATA CAC CAT CCA TC
E. coli
D22-2 (pSL106)
S. Levy (20)
tetE
F, AAA CCA CAT CCT CCA TAC GC; R, AAA TAG
GCC ACA ACC GTC AG
E. coli
D22-14 (pSL1504)
S. Levy (19)
tetG
F, CAG CTT TCG GAT TCT TAC GG; R, GAT TGG
TGA GGC TCG TTA GC
E. coli
HB101 (pJA8122)
T. Aoki (37)
tetH
F, CCT GAA AAC CAA ACT GCC TC; R, ACA GAC
CAT CCC AAT AAG CG
Pasteurella multocida
(pVM112)
M. Roberts (15)
catI
F, TCA GCT GGA TAT TAC GGC CT; R, CAT TCT
GCC GAC ATG GAA G
LK 169 (pBR329)
2
catII
F, ATT CAG CCT GAC CAC CAA AC; R, CTT CCT
GCT GAA ACT TTG CC
E. coli
J52 (pSA)
M. Roberts (25)
catIII
F, CCC ACA ATT CAC CGT ATT CC; R, GAA CCT
GTA CTG AGA GCG GC
E. coli
J53 (R387)
M. Roberts (24)
sulI
F, CAC CGC GGC GAT CGA AAT GC; R, GGT TTC
CGA GAA GGT GAT
820
Proteus mirabilis
P. H. Roy (18)
sulII
F, ATC GCT CAT CAT TTT CGG CA; R, CTC GTG
TGT GCG CAT GAA GT
Serovar Typhimurium CO-8861
C. Clark (31)
DhfrI
F, CGA AGA ATG GAG TTA TCG GG; R, TAA ACA
TCA CCT TCC GGC TC
C600 (R483)
32
aadA1
F, GCG CTA AAT GAA ACC TTA AC; R, TCG CCT
TTC ACG TAG TGG AC
E. coli
JE 2571 (pHH1457)
D. Taylor (9)
aadA2
F, TGT TGG TTA CTG TGG CCG TA; R, GCT GCG
AGT TCC ATA GCT TC
Serovar Typhimurium PT104 96-5227
D. Taylor (7)
aph3
⬘
Ia
F, TTA TGC CTC TTC CGA CCA TC; R, GAG AAA
ACT CAC CGA GGC AG
E. coli
JE 2571 (pHH1457)
D. Taylor (9)
aac6
⬘
Iq
F, GCT GGA AAT GAA TCA TGG GT; R, TAA TTC
CCC TAC CCT TCG CT
BR-SA-97-368
D. Rodrigues
(23)
bla
TEM-1F, ATA AAA TTC TTG AAG ACG AAA; R, GAC AGT
TAC CAA TGC TTA ATC A
Neisseria gonorrhoeae
18795
14
Integron 5
⬘
CS/
3
⬘
CS
F, GGC ATC CAA GCA GCA AG; R, AAG CAG ACT
TGA CCT GA
Serovar Typhimurium PT104 96-5227
D. Taylor (18)
aF, forward primer; R, reverse primer.
bThe reference strain served as a positive control for PCRs.
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added to the outer buffer chamber. The electrophoresis unit was placed on a tray and surrounded with ice. Electrophoresis was performed in three steps: 100 V for 1 h, 250 V for another hour, and finally, 500 V for 30 min. IEF gels were then dismantled from the unit, and the glass plates were separated while leaving the gel on one glass plate. To visualize beta-lactamase activity, 1 ml of nitrocefin stock solution (1 mg/ml) was added to 6 ml of molten 3% agarose in 50 mM phosphate buffer (pH 7.5) (cooled to 50 to 60°C), mixed by inversion, and then poured evenly over the gel. The presence of pink/red lines on the gel indicated beta-lactamase activity. Pictures of IEF gels were taken using a dark green filter, and the gels were transilluminated with white light.
Detection of antimicrobial resistance genes.PCR was used to detect antimi-crobial resistance genes and the presence of integrons in 11 isolates resistant to (at least) the following antimicrobials: ampicillin, chloramphenicol, streptomy-cin, sulfamethoxazole-trimethoprim, and tetracycline. Most of the primers used for the characterization of pentaresistant Salmonella serovar Typhimurium DT104 were previously described (27), and are all listed in Table 1. The DNA from the reference strains (also listed in Table 1) served as positive controls for the PCRs. Negative controls for PCRs consisted of all the reagents used for each primer pair minus the DNA template. Genomic DNA from cultures grown at 35°C on Mueller-Hinton agar with antimicrobials was extracted with a Puregene kit (Gentra Systems, Inc., Minneapolis, MN). The PCR mix for the detection of resistance genes and integrons included 1.0M of forward and reverse primers, 1⫻Taqpolymerase buffer, 1.5 mM MgCl2, 200M of each deoxynucleotide (dATP, dCTP, dGTP, and dTTP) (Gibco BRL, Burlington, Ontario), 0.025 U/l
Taqpolymerase (Gibco BRL, Burlington, Ontario), and approximately 1g of template DNA. Amplification conditions for all of the PCRs, except for integron andblaTEMamplification, were 1 cycle at 94°C for 5 min and 35 cycles for 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 s. An annealing temperature of 48°C was used for the amplification ofblaTEM. Integron amplification involved 1 cycle at 94°C for 12 min and 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 5 min. PCR products were analyzed by gel electrophoresis in a 1% agarose
gel run at 100 V for 1 h. To visualize band migration, the gel was stained with ethidium bromide and observed under UV light. A 100-bp or 1-kb ladder (Gibco BRL, Ontario) was used to estimate amplicon size.
DNA sequencing.Amplicons resulting from PCRs using the primers specific to the 5⬘conserved and 3⬘semiconserved segments or universalblaTEMprimers were sequenced in both directions using an ABI Prism 377 DNA sequencer (Applied Biosystems Division of Perkin-Elmer, Foster City, CA). DNA se-quences were compared to those in the GenBank database (National Center for Biotechnology Information) by using the BLAST suite of sequence similarity-searching programs (3, 4).
Pulsed-field gel electrophoresis (PFGE). Genomic DNA was prepared as described previously by Persing et al. (29) with modifications. Serovar Infantis strains were grown in 10 ml of Mueller-Hinton broth at 37°C for 12 to 18 h. Cells were harvested by centrifugation at 2,000⫻g for 15 min. After discarding the supernatant, cells were resuspended with 1 ml of sterilized saline (0.85% NaCl) and the concentration was adjusted to 1⫻106
cells/ml. A 5-l aliquot of cell suspension was added to 300l of TEN buffer (0.5 M EDTA, 1 M Tris base, 4 M NaCl, pH 7.5) before embedding it in 340l of low-melting-point agarose (Sigma-Aldrich Corporation, St. Louis, MS). Plugs were subjected to lysis for 5 h at 37°C in EC buffer (0.5 M EDTA, 1 M Tris base, NaCl, N-lauryl sarcosyl, Brij 58, sodium deoxycholate, pH 7.0) (Sigma-Aldrich, MS). RNase (10 mg/ml) (Sigma-Aldrich, MS) was added to the plugs for an overnight incubation at 37°C, and then proteinase K (20 mg/ml; Gibco BRL) treatment of the plugs was performed for 24 h at 54°C. Serovar Infantis strain plugs were washed four times with CHEF-TE 1⫻buffer (0.5 M EDTA, 1 M Tris base, pH 7.5) (Sigma-Aldrich, MS), followed by four washes with DNS buffer (1 M Tris base, 1 M MgCl2) (Sigma-Aldrich, MS). The digestion step was performed for 20 h at 37°C with the restriction endonuclease SpeI (10 U/l) (Amersham Pharmacia Biotech, En-gland). Electrophoresis was performed at 6 V/cm for 22 h with switch time intervals of 0.5 to 25 s for 19 h and 30 to 60 s for 3 h on CHEF DRIII (Bio-Rad Laboratories, Richmond, CA). The agarose gels were stained with ethidium bromide, visualized by UV transillumination, and photographed on Image-Master VDS (Amersham Pharmacia Biotech, England). The fragment restriction patterns were analyzed by BioNumerics (Applied Maths, Belgium) and com-pared through the construction of a similarity matrix by using the Dice coefficient with a position tolerance setting of 1.0% and optimization setting of 1.0%, which generated a dendrogram. Serovar Branderup was included as a control. A clonal structure definition of serovar Infantis was achieved according to the criteria of Tenover et al., which correlates the number of fragment differences with genetic events (33).
Two human epidemiologically unrelated serovar Infantis strains from other public health institutions of northern (a susceptible strain from Para´) and mid-western Brazil (a multidrug-resistant strain from Brasilia) were used to assess the utility of PFGE as an epidemiological marker for nosocomial infections.
RESULTS AND DISCUSSION
[image:3.585.44.283.70.349.2]Many researchers are successfully using PFGE to investigate
the epidemiologies of strains involved in outbreaks caused by
beta-lactamase- and ESBL-producing bacteria (8). The PFGE
analysis of the 35 serovar Infantis strains resulted in five PFGE
restriction fragment profiles (Fig. 1 and 2). The comparative
evaluation of the PFGE profiles yielded four fragment patterns
(A1, A3, A4, and A5) for HA isolates. Three HB and five HC
FIG. 1. PFGE: macro restriction fragment patterns of
Salmonella
serovar Infantis genome digested with SpeI. Lanes: M, molecular
weight marker of
Salmonella
Branderup strains; A2, PFGE profile of
3 HD strains; C, PFGE profile of a midwestern hospital strain; B,
PFGE profile of a northern hospital strain; A1, PFGE profile of 18
HA, 4 HB, 5 HC strains; A3, PFGE profile of 1 HA strain; A5, PFGE
profile of 1 HA strain; A4, PFGE profile of 1 HA strain.
FIG. 2. PFGE macro-restriction fragment polymorphism.
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[image:3.585.300.542.71.181.2]isolates had the PFGE profile A1 that was also encountered in
18 HA strains. The three HD strains (PFGE profile A2),
iso-lated in 2001, showed 95% similarity to PFGE profiles A1 and
A3 (Fig. 1). Macro-restriction fragment patterns of strains
from northern (PFGE profile B) and midwestern (PFGE
pro-file C) regions of Brazil were completely different from those
of strains from Rio de Janeiro, Brazil.
The A1 profile, found in 26 strains isolated from 1996 to
2001 in HA, HB, and HC, was considered to be the PFGE
profile associated with the MDR serovar Infantis outbreaks.
The PFGE patterns of serovar Infantis strains were then
clas-sified according to their similarities to the outbreak pattern.
Patterns that differed from the outbreak pattern by two
frag-ments (
ⱕ
90%) were considered to be subtypes. A variation of
two to three fragments in a PFGE profile can occur when
strains are cultured repeatedly or isolated multiple times from
the same patient (33). Those patterns that differed by at least
four fragments were classified as unrelated types by
consider-ing that they derived from two genetic events and their
isolat-ing origins.
The susceptibility profiles of serovar Infantis are shown in
Table 2. All of the strains were susceptible to carbapenem
(imipenem), ciprofloxacin, nalidixic acid, and cephamycin
(cefoxitin). All of the strains, except for one, were resistant to
ampicillin, and most were resistant to cephalosporins
(includ-ing extended spectrum). It is interest(includ-ing that strains resistant
to the highest number of antimicrobials (resistance profile
ACSSuTTmKG, etc. [Table 2]) had similar PFGE profiles and
were isolated from 1996 to 2001 from patients in different
hospitals. The high prevalence of resistance to these particular
antimicrobials may be due to selective pressure since these
antimicrobials, with the exception of kanamycin and
strepto-mycin, are among the agents most often prescribed in these
hospitals. Resistance to kanamycin and streptomycin, however,
may have been acquired through horizontal gene transfer since
aminoglycoside resistance genes are often found on plasmids
and transposons that encode resistance determinants for other
classes of antimicrobials (34, 36). Tetracycline resistance
(97.2%) and aztreonam resistance (96.1%) were also common
among the multidrug-resistant strains. It is not surprising that
the four hospitals involved in this study experienced great
difficulties in deciding which antimicrobials to use for
treat-ment. The implementation of effective screening methods for
the detection of beta-lactamases and ESBLs as well as the
establishment of surveillance programs became key factors in
the control of hospital outbreaks (16).
PCR detection of resistance genes in nine isolates resistant
to five classes of antimicrobials, represented by ampicillin,
chloramphenicol, streptomycin, sulfamethoxazole, and
tetracy-cline, showed that all of the strains with the ACSSuTTmKG
resistance profile carried
bla
TEM,
catI
,
aadA1
,
sulI
,
sulII
, and
[image:4.585.47.541.90.370.2]tet
(D) resistance genes and an integron containing an
aac
(
6
⬘
)
-Iq
gene cassette that codes for amikacin resistance
(Table 3). The only variation among these strains was the
TABLE 2. Antimicrobial resistance and PFGE profiles for serovar Infantis strains isolated between 1996 and 2001
from four Brazilian hospitals
Resistance profile(s)a PFGE
profile
No. of strains
Yr of
isolation Hospital
b
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
1
1996
HA
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
4
1996
HC
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
2
1997
HA
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
1
1998
HA
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
4
1999
HA
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
1
1999
HB
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A2
1
2001
HD
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, FEP)
A1
1
1997
HB
ACSSuTTmKG (ATM, CEF, CXM, CTX, CRO, FEP)
A1
1
1997
HA
ACSSuTTmG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
1
1998
HA
ACSSuTTmG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A2
1
2001
HD
ASSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
1
1997
HA
ACSuTTm (ATM, CEF, CXM, CTX, CRO, FEP)
A1
1
1998
HA
ACSuTTm (ATM, CEF, CXM, CTX, CRO, FEP)
A4
1
1999
HA
ACSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A5
1
1998
HA
ACSuTTmG (ATM, CEF, CXM, CTX, CRO, FEP)
A1
1
1991
HA
ASTKG (ATM, CEF, CXM, CAZ, FEP)
A2
1
2001
HD
ACT (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A1
1
1999
HB
ACT (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
A3
1
1999
HA
ACT (ATM, CEF, CXM, CTX, CRO, FEP)
A1
1
1996
HC
ASTG (ATM, CEF, CAZ, FEP)
A1
1
1996
HA
ASTG (ATM, CEF, CXM, CAZ, FEP)
A1
1
1996
HA
AST (ATM, CEF, CXM, CTX, CRO, FEP)
A1
1
1999
HA
AT (ATM, CEF, CXM, CTX, CRO, FEP)
A1
1
1999
HA
AT (CEF, CXM, CTX, CRO, FEP)
A5
1
1997
HA
ASTK
A1
1
2000
HA
ACSG (CEF)
C
1
1998
PHL
Susceptible
B
1
1997
IEC
a
Cephalosporin and aztreonam resistance profiles are shown in parentheses. A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulfamethoxazole; T, tetracycline; Tm, trimethoprim; K, kanamycin; G, gentamicin.
b
PHL, Public Health Laboratory (Brası´lia, Brazil); IEC, Evandro Chagas Institute (Para, Brazil).
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presence or absence of the streptomycin/spectinomycin
resis-tance gene
aadA2
, also known as
ant
(
3
⬘
)
Ib
, a variant of the
gene
aadA1
[
ant
(
3
⬘
)
Ia
]. The serovar Infantis strain with the
ACSuTTmKG resistance profile was intermediately resistant
to streptomycin and yet carried both
aadA1
and
aadA2
.
Re-dundancy of resistance genes was also detected in 10 strains
carrying two sulfonamide resistance genes,
sulI
and
sulII
.
Thirty-two (91.4%) serovar Infantis strains were classified as
clavulanic-acid-inhibited ESBL-producing strains according to
CLSI standards (21 from HA, 3 from HB, 5 from HC, and 3
from HD). Twenty-two strains (62.8%) were resistant to both
CTX and CAZ (Table 2), which suggested the presence of at
least one ESBL. According to the beta-lactamase classification
scheme of Bush et al. (1995), cefotaximases are class A ESBLs
(group 2be) that generally have higher hydrolytic activities
against cefotaxime than ceftazidime, while ceftazidimases (also
group 2be ESBLs) generally hydrolyze ceftazidime more
readily than cefotaxime (10). In addition, group 2be ESBLs
inactivate not only extended-spectrum cephalosporins but also
monobactams such as aztreonam. Ten of the serovar Infantis
strains characterized in this study were resistant to both CTX
and CAZ, while only one was resistant to only CTX (Table 3).
DNA sequencing of the amplicons obtained with
bla
TEMprim-ers (which targeted the conserved region of TEM-related
en-zymes) revealed the presence of the non-ESBL
bla
TEM-1. In
order to determine whether more than one beta-lactamase was
produced by these 11 multidrug-resistant serovar Infantis
strains, isoelectric focusing was performed (Table 4).
The pI profiles indicated the presence of beta-lactamases
with pI values of 5.4, 6.3, 6.9, and 9.0. The six strains with the
antibiogram ACSSuTTmKG (resistance profile, ATM, CEP,
CXM, CAZ, CTX, CRO, FEP) had at least four different
lactamases (since there could be more than one
beta-lactamase present in a strain with the same pI value), while
another strain with the same resistance profile produced only
two types of beta-lactamases (pIs 9.0 and 5.4). This result is
significant since all seven strains are resistant to CEP, CXM,
CAZ, CTX, CRO, and FEP, indicating that resistance to those
cephalosporins requires the presence of only two types of
beta-lactamases with pI values of 5.4 and 9.0. In addition, those
seven strains are also resistant to the monobactam ATM,
which indicates, according to Bush et al., that a group 2be
ESBL is present within the strain (10).
The presence of identical antimicrobial resistance genes and
the close relatedness of strains as determined by PFGE
anal-ysis provides evidence that the hospitals involved in this study
had a salmonellosis outbreak that was caused by serovar
In-fantis strains that shared the same phylogenetic lineage. It is
important to emphasize that strains from HC were isolated in
only 1996, while strains from HB were isolated in 1997 and
1999. HA strains were isolated from 1996 to 1999. At the
beginning of 2001, HD was informed about the characteristics
and clonal nature of multidrug-resistant serovar Infantis so
that appropriate control measures could be developed and,
subsequently, serovar Infantis was no longer detected in the
hospital environment. The guidelines and rules that provide
for the planning of the National Program of Hospital Infection
Control were defined by administrative rule GM 2.616 as of 12
May, 1998. This decree categorizes children hospitalized in
high-risk nurseries as intensive-care patients requiring
partic-ular attention to infections due to multidrug-resistant
patho-gens (22). These patients are subjected to standard procedures
for controlling nosocomial infections, such as the cleaning and
disinfection of medical equipment, frequent hand washing,
patient-to-patient contact precautions, and the monitoring of
patients’ stools for the presence of multidrug-resistant serovar
Infantis. The best strategy for antimicrobial therapy and
spe-cific infection control measures for each patient was
deter-mined on a case-by-case basis (1).
[image:5.585.42.543.82.163.2]The results in this study indicate that efficient surveillance
programs and effective decontamination procedures must be
TABLE 3. Antimicrobial resistance genes detected in multidrug-resistant serovar Infantis strains
Resistance profilea No. of
strains Integron gene
b PFGE
profile Antimicrobial resistance genes c
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
6
aac(6
⬘
)-Iq
A1
bla
TEM,
catI
,
aadA1
,
sulI/II
,
tet
(D)
ACSSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
1
aac
(
6
⬘
)
-Iq
A1
bla
TEM,
catI
,
aadA1/A2
,
sulI/II
,
tet
(D)
ACSSuTTmKG (ATM, CEF, CXM, CTX, CAZ, FEP)
1
aac
(
6
⬘
)
-Iq
A1
bla
TEM,
catI
,
aadA1
,
sulI/II
,
tet
(D)
ACSSuTTmKG (ATM, CEF, CXM, CTX, CRO, FEP)
1
aac
(
6
⬘
)
-Iq
A1
bla
TEM,
catI
,
aadA1/A2
,
sulI/II
,
tet
(D)
ACSuTTmKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
1
daac
(
6
⬘
)
-Iq
A5
bla
TEM
,
catI
,
aadA1/A2
,
sulI/II
,
tet
(D)
ASSuTKG (ATM, CEF, CXM, CAZ, CTX, CRO, FEP)
1
A1
bla
TEM,
aadA1/A2
,
sulII
,
tet
(D)
aCephalosporin and aztreonam resistance profiles are shown in parentheses. A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulfamethoxazole; T, tetracycline; Tm, trimethoprim; K, kanamycin; G, gentamicin.
bThe integron cassette size was 1,269 bp.
c“aadA1/A2” or “sulI/II” indicates the presence of bothaadA1andaadA2or bothsulIandsulII, homologous genes, respectively, within a strain. dThis strain was intermediately resistant to streptomycin.
TABLE 4.

-Lactamase profiles detected in multidrug-resistant
serovar Infantis strains
Resistance profilea No. of strains
PFGE profile
Isoelectric points of-lactamases
ACSSuTTmKG (ATM, CEF, CXM,
CAZ, CTX, CRO, FEP)
6
A1
9, 6.9, 6.3, 5.4
ACSSuTTmKG (ATM, CEF, CXM,
CAZ, CTX, CRO, FEP)
1
A1
9, 5.4
ACSSuTTmKG (ATM, CEF, CXM,
CTX, CRO, FEP)
1
A1
9, 6.9, 6.3, 5.4
ACSSuTTmKG (ATM, CEF, CXM,
CAZ, CTX, FEP)
1
A1
9, 6.9, 6.3, 5.4
ACSuTTmKG (ATM, CEF, CXM,
CAZ, CTX, CRO, FEP)
1
A5
9, 6.3, 5.4
ASSuTKG (ATM, CEF, CXM,
CAZ, CTX, CRO, FEP)
1
A1
9, 6.9, 5.4
aCephalosporin and aztreonam resistance profiles are shown in parentheses. A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulfamethoxazole; T, tetracycline; Tm, trimethoprim; K, kanamycin; G, gentamicin.
on May 16, 2020 by guest
http://jcm.asm.org/
[image:5.585.301.540.560.698.2]implemented for the prevention of nosocomial outbreaks of
salmonellosis caused by multidrug-resistant serovar Infantis.
ACKNOWLEDGMENTS
We thank C. M. F. Reis and A. F. M. Santos (FIOCRUZ, Rio de
Janeiro, Brazil) for her collaboration on the PFGE technique and
photo documentation and E. Soares and his working group
(FIOCRUZ, Rio de Janeiro, Brazil), who provided assistance and
supplied reagents.
This work was supported by grants from the Oswaldo Cruz Institute
Pos-Graduation/FIOCRUZ-Rio de Janeiro and National Council for
Sci-entific and Technological Development (CNPq), Brazil. O. Mykytczuk’s
student stipend was from the National Microbiology Laboratory and the
University of Manitoba, Winnipeg, Manitoba, Canada.
REFERENCES
1.Ageˆncia Nacional de Vigilaˆncia Sanita´ria (ANVISA).2005. Manual de pro-cedimentos ba´sicos em microbiologia clı´nica para o controle de infecc¸a˜o hospitalar. [Online.] http://www.hu.usp.br/arquivos/Manualccih_2005.pdf. 2.Alton, N. K., and D. Vapnek.1979. Nucleotide sequence analysis of the
chloramphenicol resistance transposon Tn9. Nature282:864–869. 3.Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman.1990.
Basic local alignment search tool. J. Mol. Biol.215:403–410.
4.Altschul, S. F., T. L. Madden, A. A. Scha¨ffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman.1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res.25:3389–3402. 5.Angulo, F. J., K. R. Johnson, R. V. Tauxe, and M. L. Cohen.2000. Origins
and consequences of antimicrobial-resistant nontyphoidalSalmonella: impli-cations for the use of fluoroquinolones in food animals. Microb. Drug Resist. 6:77–83.
6.Asensi, M. D., and E. Hofer.1994. Serovars and multiple drug resistant
Salmonellasp. isolated from children in Rio de Janeiro – Brazil. Rev. Mi-crobiol. Sa˜o Paulo25:149–153.
7.Bito, A., and M. Susani.1994. Revised analysis ofaadA2gene of plasmid pSa. Antimicrob. Agents Chemother.38:1172–1175.
8.Bradford, P. A.2001. Extended-spectrum beta-lactamase in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev.14:933–951.
9.Bradley, D. E., D. E. Taylor, and D. R. Cohen.1980. Specification of surface mating systems among conjugative drug resistance plasmids inEscherichia coliK-12. J. Bacteriol.143:1400–1470.
10.Bush, K., G. A. Jacoby, and A. A. Medeiros.1995. A functional classification scheme for-lactamases and its correlation with molecular structure. Anti-microb. Agents Chemother.39:1211–1233.
11.Chiappini, E. L., L. Galli. P. Pecile, A. Vierucci, and M. de Martino.2002. Results of a 5-year prospective surveillance study of antibiotic resistance amongSalmonellaenterica isolates and ceftriaxone therapy among children hospitalized for acute diarrhea. Clin. Ther.24:1585–1594.
12.Costa, G. A., and E. Hofer.1972. Isolamento e identificac¸a˜o de Enterobac-te´rias, p. 120. Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, Brazil. 13.De Moraes, B. A., C. A. N. Cravo, M. M. Loureiro, C. A. Solari, and M. D. Asensi.2000. Epidemiological analysis of bacterial strains involved in hos-pital infection in a university hoshos-pital from Brazil. Rev. Inst. Med. Trop. Sa˜o Paulo42:201–207.
14.Edelstain, M., and L. Stratchounski.1998. Development of single-strand conformational polymorphism (SSCP) PCR method for discriminatory de-tection of genes coding for TEM-family-lactamases. Program Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., poster E-96.
15.Hansen, L. M., L. M. McMurry, S. B. Levy, and D. C. Hirsh.1993. A new tetracycline resistance determinant, Tet H, fromPasteurella multocida spec-ifying active efflux of tetracycline. Antimicrob. Agents Chemother.37:2699– 2705.
16.Kaye, K. S., H. S. Fraimow, and E. Abrutyn.2000. Pathogens resistant to antimicrobial agents—epidemiology, molecular mechanisms and clinical management. Infect. Dis. Clin. N. Am.14:293–317.
17. Reference deleted.
18.Le´ve`sque, C., L. Piche´, C. Larose, and P. H. Roy.1995. PCR mapping of
integrons reveals several novel combinations of resistance genes. Antimi-crob. Agents Chemother.39:185–191.
19.Marshall, B., S. Morrissey, P. Flynn, and S. B. Levy.1986. A new tetracy-cline-resistant determinant, class E, isolated fromEnterobacteriaceae. Gene 50:111–117.
20.Marshall, B., C. Tachibana, and S. B. Levy.1983. Frequency of tetracycline resistance determinant classes among lactose-fermenting coliforms. Antimi-crob. Agents Chemother.24:835–840.
21.Mendez, B., C. Tachibana, and S. B. Levy.1980. Heterogeneity of tetracy-cline resistance determinants. Plasmid3:99–108.
22.Ministe´rio da Sau´de.1998. Portaria n° 2.616, de 12 de maio de 1998. Dispo˜e sobre a obrigatoriedade da manutenc¸a˜o do “Programa Nacional de Controle de Infecc¸a˜o Hospitalar.” Ministe´rio da Sau´de, Brazilia, Brazil.
23.Mulvey, M. R., D. A. Boyd, L. Baker, O. Mykytczuk, E. M. Reis, M. D. Asensi, D. P. Rodrigues, and L. K. Ng.2004. Characterization of a Salmonella entericaserovar Agona strain harbouring a class 1 integron containing novel OXA-type -lactamase (blaOXA-53) and 6⬘-N-aminoglycoside acetyltrans-ferase genes [aac(6⬘)-I30]. J. Antimicrob. Chemother.54:354–359. 24.Murray, I. A., J. V. Martinez-Suarez, T. J. Close, and W. V. Shaw.1990.
Nucleotide sequences of genes encoding the type II chloramphenicol acetyl-transferases ofEscherichia coliandHaemophilus influenzae, which are sen-sitive to inhibition by thiol-reactive reagents. Biochem. J.272:505–510. 25.Murray, I. A., A. R. Hawkins, J. W. Keyte, and W. V. Shaw.1988. Nucleotide
sequence analysis of overexpression of the gene encoding a type III chlor-amphenicol acetyltransferase. Biochem. J.252:173–179.
26.NCCLS.2004. Performance standards for antimicrobial susceptibility test-ing; 30th informational supplement. NCCLS document no. M100–S14, vol. 24. NCCLS, Wayne, Pa.
27.Ng, L.-K., M. R. Mulvey, I. Martin, G. A. Peters, and W. Johnson.1999. Genetic characterization of antimicrobial resistance in Canadian isolates of
Salmonellaserovar Typhimurium DT104. Antimicrob. Agents Chemother. 43:3018–3021.
28.Olsen, S. J., R. Bishop, F. W. Brenner, T. H. Roels, N. Bean, R. V. Tauxe, and L. Slutsker.2001. The changing epidemiology ofSalmonella: trends in se-rotype isolated from humans in the United States, 1987-1997. J. Infect. Dis. 183:753–761.
29.Persing, D. H., T. F. Smith, F. C. Tenover, and T. J. White (ed.).1993. Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, Washington, D.C.
29a.Poppof, M. Y.2001. Antigenic formulas of theSalmonellaserovars. WHO Collaborating Center for Reference and Research for Salmonella Serovars, Institut Pasteur, Paris, France.
30.Pessoa-Silva, C. L., C. M. Toscano, B. M. Moreira, A. L. Santos, A. C. Frota, C. A. Solari, E. L. Amorim, M. da Gloria S. Carvalho, L. M. Teixeira, and W. R. Jarvis.2002. Infection due to extended-spectrum beta-lactamase-producingSalmonella entericasubsp.entericaserovar Infantis in a neonatal unit. J. Pediatr.141:381–387.
31.Rådstro¨m, P., and G. Swedberg.1988. RSF1010 and a conjugative plasmid containsulII, one of two known genes for plasmid-borne sulfonamide resis-tance dihydropteroate synthase. Antimicrob. Agents Chemother.32:1684– 1692.
32.Sundstro¨m, L., and O. Sko¨ld.1990. ThedhfrItrimethoprim resistance gene can be found at specific sites in other genetic surroundings. Antimicrob. Agents Chemother.34:642–650.
33.Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan.1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol.33:2233–2239.
34.Tolmasky, M. E., R. M. Chamorro, J. H. Crosa, and P. M. Marini.1988. Transposon-mediated amikacin resistance inKlebsiella pneumoniae. Antimi-crob. Agents Chemother.32:1416–1420.
35.Winokur, P. L., A. Brueggemann, D. L. DeSalvo, L. Hoffmann, M. D. Apley, E. K. Uhlenhopp, M. A. Pfaller, and G. V. Doern.2000. Animal and human multidrug-resistant, cephalosporin-resistantSalmonellaisolates expressing a plasmid-mediated CMY-2 AmpC beta-lactamase. Antimicrob. Agents Che-mother.44:2777–2783.
36.Vakulenko, S. B., and S. Mobashery.2003. Versatility of aminoglycosides and prospects for their future. Clin. Microbiol. Rev.16:430–450. 37.Zhao, J., and T. Aoki.1992. Nucleotide sequence analysis of the class G
tetracycline resistance determinant fromVibrio anguillarum. Microbiol. Im-munol.36:1051–1060.
