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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, October 2000, p. 2911-2914, Vol. 44, No. 10
Laboratory of Bacteriology and Medical
Mycology1 and Laboratory of Cellular
Biology,2 Istituto Superiore di Sanità,
Dipartimento di Biologia, Università di Roma
Tre,3 and IRCCS Lazzaro
Spallanzani,4 Rome, Italy
Received 17 February 2000/Returned for modification 18 May
2000/Accepted 1 July 2000
A conjugative IncL/M plasmid (pSEM) conferring resistance to
gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella
enterica serotype Typhimurium. Resistance to aminoglycosides was
encoded by a sul1-type class 1 integron (In-t3). An
extended-spectrum beta-lactamase gene,
blaSHV-5, was identified 3.5 kb downstream of
the integrase (intI1) gene of In-t3. Nucleotide sequence
analysis of the 5.3-kb blaSHV-5-In-t3 region
of pSEM highlighted striking similarities with IncL/M plasmids isolated
from nosocomial gram-negative pathogens, conferring resistance to
expanded-spectrum cephalosporins and aminoglycosides.
Although antibiotic resistance is
becoming a major threat to human health worldwide, information
concerning the dissemination and geographical distribution of
antibiotic-resistant bacterial pathogens remains scattered (8, 14,
26). During the last decade many hospital outbreaks caused by
extended-spectrum beta-lactamase (ESBL)-producing
Enterobacteriaceae have been reported, and most of the
ESBL-producing strains were found to carry the
blaTEM-1, blaTEM-2, and
blaSHV-1 gene derivatives (5, 6, 9, 11, 19,
27). These genes are prevalently located on large conjugative plasmids of the incompatibility (Inc) groups IncC, IncFI, IncH12, and
IncL/M (3, 5, 7, 13, 18, 20, 23, 28).
More recent reports have highlighted the emergence of ESBL-producing
strains endowed with an extremely wide spectrum of antibiotic resistance, including resistance to sulfonamides, trimethoprim, streptomycin, kanamycin, gentamicin, and amikacin (17, 21, 23). However, the molecular mechanisms involved in the
acquisition and/or transmission of ESBL-encoding genes are poorly understood.
In previous work we identified an IncL/M plasmid, referred to as pSEM,
in eight epidemiologically unrelated, multiple-drug-resistant strains
of Salmonella enterica serotype Typhimurium. This plasmid contains a sul1-type class 1 integron (In-t3) carrying the
aacA4, aacC1, and aadA1 gene cassettes
and conferring resistance to sulfonamides, kanamycin, gentamicin, and
amikacin (29). The streptomycin and spectinomycin-resistance
determinant, aadA1, is poorly expressed in In-t3 because it
is located in a distal position relative to the main integron promoter
Pant (29).
The Salmonella strains harboring pSEM were analyzed in more
detail with regard to their antibiotic resistance profile.
Microdilution susceptibility tests demonstrated that all eight strains
were resistant to ceftazidime ( To clone the gene responsible for resistance to expanded-spectrum
cephalosporins, an EcoRI genomic library was constructed in
the pUC18 vector using total DNA extracted from the prototypic Salmonella 202 strain. The library was introduced by
transformation in Escherichia coli DH5 Conjugal transfer of pSEM from S. enterica serotype
Typhimurium strain 202 to E. coli CSH26R (29) was
associated with transmission of the ceftazidime-resistance determinant,
providing direct genetic evidence of the physical link between pSEM and
the blaSHV-5 gene. pSEM was purified from the
ceftazidime-resistant E. coli 202-24 exconjugant, digested
with EcoRI or with XbaI (Fig.
1A), and analyzed by Southern blot
hybridization with the blaSHV-5 and
int1I gene probes (Fig. 1B). The
blaSHV-5 and int1I probes recognized
two different EcoRI bands of 13.5 and 12.5 kb, respectively.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Multiple-Antibiotic Resistance Mediated by Structurally
Related IncL/M Plasmids Carrying an Extended-Spectrum
-Lactamase Gene and a Class 1 Integron
ABSTRACT
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16 µg/ml), cefotaxime (32
µg/ml), ceftriaxone (64 µg/ml), cefamandole (32 µg/ml),
cefoxitin (8 µg/ml), and aztreonam (16 µg/ml) but sensitive to
imipenem (<4 µg/ml).
competent cells,
and selection was performed on Luria-Bertani agar plates containing
ceftazidime (4 µg/ml). Plasmids extracted from six independent
ceftazidime-resistant transformants contained a 13.5-kb
EcoRI insert. The DNA of one randomly selected recombinant
plasmid, designated pE135, was used as the template in PCR
amplification experiments performed with OS5-OS6 or TEMA-TEMB primer
pairs specifically designed to amplify the
blaSHV-1 and blaTEM-1
gene derivatives, respectively (2, 15). An amplicon of 795 bp was obtained with the OS5-OS6 primer pair, and its complete sequence
unambiguously identified the blaSHV-5 gene
(1, 12) (sequence accession no. P37320).
View larger version (53K):
[in a new window]
FIG. 1.
(A) agarose gel electrophoresis of XbaI- and
EcoRI-digested pSEM extracted from E. coli
202-24. HindIII-digested lambda DNA
( 
-HindIII) and 1-kb marker (KiloBase DNA marker;
Pharmacia Biotech, Milan, Italy) were used as standards. (B) Southern
blot hybridization (25) of EcoRI-digested pSEM
(electrophoresed as in panel A) with
[-32P]dCTP-labeled blaSHV-5 and
intI1 probes. The blaSHV-5 probe was
obtained by PCR amplification with the OS5-OS6 primer pair. The
intI1 probe was obtained by agarose elution of the 596-bp
PvuII-RsaI restriction fragment from the plasmid
pACYC184::Tn21 (4).
To characterize the blaSHV-5 flanking regions
and locate the blaSHV-5 gene relative to the
In-t3 position on pSEM, extended PCR amplification experiments were
performed on total DNA extracted from S. enterica serotype
Typhimurium 202, with the aacA4R-OS5 or aacA4R-OS6 primer pairs. An
amplicon of approximately 5.5 kb was obtained only with the aacA4R-OS6
primer pair, indicating that the blaSHV-5 gene
was located approximately 3.5 kb downstream of the int1I
gene of In-t3. The amplicon was digested with SpeI and
BamHI for ligation in the pT-Adv vector, yielding plasmids pTASpe1, pTASpe2, and pTABam. Further subclones of the pTASpe2 construct were obtained by EcoRV and KpnI
internal deletions (24). The DNA inserts of these subclones
and the blaSHV-5-flanking regions in pE135 were
entirely sequenced (25). A consensus map of pSEM, encompassing the In-t3 integron and the blaSHV-5
gene, is shown in Fig. 2.
|
Nucleotide sequence comparisons of the 4,379-bp fragment of pTASpe2
revealed striking homologies between pSEM and promiscuous plasmids
carrying ceftazidime-resistance determinants in different gram-negative
bacteria. The 2,254-bp segment encompassing the blaSHV-5 gene matched a homologous sequence
(99.8% identity) on plasmid pPAG-KE from a clinical Pseudomonas
aeruginosa isolate (accession no. AF096930) (E. Scoulica, A. Aransay, and I. Tselentis, unpublished results, 1998). In the
blaSHV-5-preceding region, two open reading
frames (ORFs) were identified on opposite DNA strands; their predicted
translation products (277 and 297 amino acids) do not show significant
homology with known proteins. However, the pSEM region spanning from
nucleotide 2654 to 3456 was 81% identical to the Klebsiella
pneumoniae lacY gene (from nucleotide 416 to 1215 of the sequence
released under accession no. X14154), encoding the lactose transport
protein. A deletion of the first 130 codons and an opale
mutation at codon 174 predicted the deduced translation product to be
nonfunctional, justifying the cognate gene designation
lacY. Interestingly, an IS26 element was found juxtaposed to codon 131 of the lacY pseudogene in the
HindIII-SpeI region of the pSEM. This
element, which also includes the transposase gene (tnpA) and
the inverted repeats (16), is flanked by the 5' conserved
segment (5'CS) region of In-t3 with a 113-bp deletion (5'CS)
(10). The IS26 element of pSEM is identical to
the IS26 element flanking the
blaSHV-2 gene in plasmids pPa-1 from
P. aeruginosa and pZMP1 from K. pneumoniae
(accession no. AF074954 and X53817, respectively). The location
of the blaSHV gene and of the
IS26 element differs between pSEM and pPa-1 and pZMP1. In
pPa-1 and pZMP1 the IS26 shortly precedes the ATG codon of
the blaSHV-2 gene, while in pSEM it is located
2,638 bp upstream of the 5' end of the blaSHV-5
gene. Insertion sequences closely linked to ESBL genes were previously
mapped on other plasmids. This was the case with pCFF04, an IncL/M
replicon of 85 kb isolated from K. pneumoniae in France,
carrying an IS15 element and the
blaTEM-3 gene on a large EcoRI band
(17). An IS6100 element was recently described in
pACM1, found in Klebsiella oxytoca isolates responsible for
a nosocomial outbreak in the United States. This IncL/M plasmid carries
the blaSHV-5 gene and a class 1 integron,
thereby displaying structural and functional similarity to pSEM
(21, 22). Remarkably, both integrons contain the same
aacA4, aacC1, and aadA1 gene cassettes and two additional ORFs, ORF X and ORF X', coding for unknown functions. A unique feature of the class 1 integrons of pSEM and pACM1
is the lack of the conserved BamHI site in the region
downstream of the intI1 gene (21). Although the
presence of integrons was not described for other IncL/M plasmids, both
aadA1 and aacA4 genes were detected by Southern
blot hybridization on pCFF04 (17). Uncharacterized genetic
determinants for gentamicin and tobramycin resistance were associated
with the blaSHV-5 gene in pIBK1, an IncL/M
conjugative plasmid of 80 kb isolated at the Innsbruck University
Hospital (Innsbruck, Austria) from K. pneumoniae strains responsible for outbreaks in intensive care units (23).
The finding that pSEM shares common traits with other ESBL-encoding plasmids is also supported by the observation that the EcoRI restriction profile of pSEM is similar to the published EcoRI patterns of pACM1, pCFF04, and pIBK1 IncL/M plasmids (17, 21, 23). The largest EcoRI fragment, which appeared to be the most variable in length (ranging from 13.5 to approximately 20 kb), was reported to contain the ceftazidime resistance determinants in all three plasmids (17, 21, 23). Thus, plasmids pSEM, pPAG-KE, pZMP1, pPa-1, pACM1, pIBK1, and pCFF04 could be members of a family of broad-host-range replicons responsible for resistance to expanded-spectrum cephalosporins in gram-negative pathogens. Common features of these plasmids are the presence of ESBL-encoding genes (SHV or TEM type), likely associated with a sul1-type class 1 integron conferring resistance to aminoglycosides and possibly the IncL/M ori/rep functions. The physical association between insertion sequences and ESBL genes in these plasmids suggests that IS elements could be involved in the assembly of antibiotic resistance islands by gene mobilization. A simple way to generate the gene configuration shown in Fig. 2 is for IS26 to have jumped either into the 5'CS of In-t3 or, from a position adjacent to it, to have deleted adjacent bases. IS26-mediated deletion of adjacent bases could also explain the relationship of pSEM to pZMP1 and pPa-1.
We emphasize that all the ESBL-encoding plasmids described thus far,
with the exception of pSEM, originate from bacteria responsible for
nosocomial outbreaks. The presence of a genetic relict, i.e., the
lactose transport protein pseudogene (lacY), could
indicate that pSEM originates from lactose-fermenting bacteria, thus
representing a recent acquisition for Salmonella, likely
occurring through horizontal transfer. The identification of this
family of antibiotic resistance plasmids in enteric bacteria
responsible for food-borne and community-acquired infections, such as
S. enterica serotype Typhimurium, has serious public health
implications. It is of concern that broad-host-range plasmids carrying
resistance determinants for a number of clinically relevant
new-generation antibiotics are spreading worldwide among bacteria
responsible for both nosocomial and community-acquired infections.
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ACKNOWLEDGMENTS |
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We are grateful to I. Luzzi, A. Pantosti, and A. Cassone for critical reading of the manuscript and to the anonymous reviewers for their constructive criticism. We also thank F. Riccobono for DNA sequencing, and S. Mariotti, I. Benedetti, and S. Arena for excellent technical assistance.
Funding for this work was provided through grants from the Ministero della Sanità, Programmi per la Ricerca Finalizzata 1998 and 1999.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy. Phone: 39-06-4990-3128. Fax: 39-06-4938-7112. E-mail: alecara{at}iss.it.
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