JAC
Journal of Antimicrobial Chemotherapy (2004) 54, 354–359 DOI: 10.1093/jac/dkh347 Advance Access publication 1 July 2004
Characterization of a Salmonella enterica serovar Agona strain harbouring a class 1 integron containing novel OXA-type b-lactamase (blaOXA-53) and 60-N-aminoglycoside acetyltransferase genes [aac(60)-I30 ] Michael R. Mulvey1*, David A. Boyd1, Lorea Baker1, Oksana Mykytczuk1, E. M. F. Reis2, M. D. Asensi2, D. P. Rodrigues2 and Lai-King Ng1 1
Received 30 April 2004; returned 29 May 2004; revised 1 June 2004; accepted 2 June 2004
Objective: To characterize by molecular methods a multidrug-resistant Salmonella enterica serovar Agona (S. enterica Agona) isolated from a hospitalized patient in Rio de Janeiro, Brazil. Methods: The S. enterica Agona strain was screened by PCR and DNA sequencing for TEM, SHV and CTX-M-type b-lactamase genes, tet(A), (B), (C) and (D) tetracycline resistance genes, chloramphenicol resistance genes and class 1 integrons. Plasmid characterization was carried out by PCR and Southern hybridization analysis. PCR and PFGE were used to characterize nine other S. enterica Agona strains collected from hospitals in Rio de Janeiro. Results: The study strain was found to harbour a 105 kb plasmid, which contained catA1, blaTEM-1, a class 1 integron with two novel genes labelled blaOXA-53 and aac(60 )-I30, respectively, and an additional unidentified aminoglycoside resistance gene. A second 53 kb plasmid from the same strain contained tet(D) and blaSHV-5. OXA-53 was shown to provide reduced susceptibility to ceftazidime, and its activity was inhibited in the presence of clavulanic acid. PFGE analysis of the nine other S. enterica Agona strains revealed two clusters of related strains (78% similarity), and PCR analysis showed that all strains contained the novel integron. Conclusion: An S. enterica Agona strain was found to harbour three plasmid-encoded b-lactamases, one (OXA-53) on a novel class 1 integron that also contains a new aminoglycoside resistance gene, aac(60 )-I30. The multidrug resistance plasmids appear to have disseminated to other city hospitals via other S. enterica Agona strains. Keywords: extended-spectrum b-lactamases, plasmid-encoded resistance, hospital-acquired
Introduction Antimicrobial drug resistance is an increasing problem in Salmonella, with strains more and more commonly being isolated exhibiting multidrug resistance (MDR).1 MDR strains are resistant to multiple classes of antimicrobials, including ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole, aminoglycosides, tetracyclines and fluoroquinolones. Resistance is often carried on transferable plasmids, which may harbour class 1 integrons containing drug resistance cassettes. In MDR Salmonella
enterica Typhimurium DT104 however, resistance can also be chromosomally located with all resistance genes found on a 43 kb structure called Salmonella genomic island 1 (SGI1).2 Further, the resistance genes are clustered near one end of SGI1 and located in a complex class 1 integron.3,4 Resistance to third-generation b-lactams in Salmonella has been reported worldwide and most often results from the production of plasmid-mediated Ambler class A or class C (AmpCtype) extended-spectrum b-lactamases (ESBLs).1 b-Lactamases from the TEM, SHV, CTX-M, PER and OXA families have
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*Corresponding author. Tel: +1-204-789-2133; Fax: +1-204-789-5020; E-mail:
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354 JAC vol.54 no.2 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.
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Bacterial and Enteric Diseases Program, National Microbiology Laboratory, Health Canada, 1015 Arlington St., Winnipeg, Manitoba, Canada, R3E 3R2; 2Enterobacteria Laboratory, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
Novel integron from Salmonella Agona
Materials and methods Bacterial strains S. enterica Agona strains were originally isolated in 1996 from the stools of patients from various hospitals in Rio de Janeiro, Brazil, and sent to the Enterobacteria Laboratory, Oswaldo Cruz Institute, FIOCRUZ. Subsequently, through a collaborative effort with the Pan-American Health Organization under the umbrella of the WHO, the strains were sent to the National Microbiology Laboratory, Health Canada. Strains were speciated using standard biochemical tests13 and confirmed as an Agona (4,12:f,g,s:-) by serogrouping and serotyping using the somatic (O) and flagellar (H) antigen, as described previously.14
Susceptibility to antimicrobials Antimicrobial susceptibility profiles of the strains used in this study were determined using disc diffusion following guidelines recommended by the NCCLS15 and Etest strips (AB Biodisk).
DNA methodology PCR was used to detect tetracycline-resistant genes,16 blaTEM,17 blaSHV,18 blaCTX,19 chloramphenicol resistance genes20 and class 1 integrons.21 PCR amplicons were purified using Montage PCR filters (Millipore) and plasmids were purified using Plasmid Midi Kits (Qiagen). Approx. 100 ng of plasmid DNA was used to transform electrocompetent Escherichia coli DH10B (Invitrogen) with selection on LB agar containing ampicillin (50 mg/L) or cefotaxime (10 mg/L). Plasmid DNA digested with restriction enzymes was separated on a 0.7% agarose gel using 0.5 Tris/Borate/EDTA for 16 h at 2.8 V/cm with circulating buffer. Southern hybridizations were carried out by standard methods22 with probes labelled and detected with ECL Kits (Amersham). DNA sequence was determined using BigDye Terminator Cycle Sequencing Kits (ABI) on an ABI 3100 automated sequencer and was carried out in the DNA Core Facility of the National Microbiology Laboratory. Primer MDR-1 (50 -TGATCGAAATCCGATCCTTG-30 ) and 30 -CS21 were used to amplify the entire class 1 integron cassette region, and the 2281 bp amplicon was purified and cloned into the vector pPCR-Script Cam, transformed into E. coli DH10B and the plasmid labelled pOA-2. The insert in pOA-2 was sequenced using vector-specific primers T7 and T3, as well as primers OXA-X-DN1 (50 -CAATCTCAGCACAGGAACAG-30 ) and AAC6X-UP1 (50 -GTTAAGATTCTAGCTGCCTG30 ). Strains were subtyped using PFGE following DNA extraction and digestion with XbaI using the standardized E. coli (O157:H7) protocol established by the CDC.23 The molecular weight standard used was XbaI-digested Salmonella enterica Braenderup ‘Universal Marker’ (kindly provided by B. Swaminathan, CDC). Isolates were considered to be genetically related if their macrorestriction DNA patterns differed by fewer than seven bands.24
Table 1. Minimum inhibitory concentrations and disc zone diameters of various antimicrobials for the strains used in this study
S. Agona 97-0017
E. coli DH10Ba
E. coli DH10B pHSN-1 [SHV-5; Tet(D)]
E. coli DH10B pHSN-2 [OXA-53, TEM-1, AAC(60 )-I30]
E. coli XL-10Gold Kanb
E. coli XL-10-Gold Kan pOA-2 [OXA-53, AAC(60 )-I30]
Etest (mg/L) cefotaxime cefotaxime/clavulanate ceftazidime ceftazidime/clavulanate amikacin gentamicin kanamycin streptomycin tobramycin
>16 >1 >32 1.5 24 >256 >256 12 >256
NDc ND ND ND 1 0.5 2 >256 0.5
256 0.5
0.25 0.38 >32 >4 16 > _ 256 > _ 256 > _ 256 96
ND ND ND ND 0.75 0.38 >256 2 0.5
32 >4 >256 1.5 >256 16 >256
Disc (diameter in mm) cefotaxime cefotaxime/clavulanate ceftazidime ceftazidime/clavulanate
ND ND ND ND
ND ND ND ND
23 40 6 30
34 34 17 24
Antimicrobial
a
Streptomycin resistant due to presence of rpsL mutation. Kanamycin resistant due to presence of Tn5. c ND, not determined. d PZ, phantom zone of inhibition. b
355
ND ND ND ND
38 39 13 24
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been described in Salmonella, as has the CMY-2 AmpC-type b-lactamase. In some cases, multiple b-lactamases from multiple families have been produced in single isolates.5,6 Salmonella enterica serovar Agona (S. enterica Agona) was first identified in Ghana.7 Since then, this serovar has been reported in many countries worldwide in both humans and animals.8 In Brazil, S. enterica Agona was the fourth most common Salmonella serotype isolated from non-human sources and was among the top 10 serotypes associated with human disease.9 Multidrug-resistant S. enterica Agona has been responsible for at least two hospital outbreaks in paediatric wards in Brazil.10,11 In both cases, the strains were found to harbour large plasmids that conferred resistance to multiple antimicrobials. Interestingly, SGI1 has also been found in S. enterica Agona strains.12 In this report, we describe an S. enterica Agona strain harbouring multiple b-lactamases, including two ESBLs, on two plasmids one of which contains a class 1 integron with a novel cassette consisting of two genes, blaOXA-53 and aac(6 0 )-I30.
M. R. Mulvey et al. Computer-assisted analysis Identity searches were conducted using the BLAST suite of programs25 and open reading frames (ORFs) detected with ORFinder, via the World Wide Web interface of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). PFGE-generated DNA profiles were digitized and entered into the BioNumerics software program version 2.5 (Applied Maths, Sint-Martens-Latem, Belgium) for analysis.
Nucleotide sequence accession number The insert in pOA-2 has been assigned accession number AY289608 in the GenBank nucleotide sequence database.
Results and discussion Characterization of the clinical isolate as an ESBL producer
Plasmid analysis To characterize further the potential mobile elements carrying the resistance genes, plasmid DNA was isolated from S. enterica Agona 97-0017 and profiling revealed the clinical isolate contained several plasmids (data not shown). Whole plasmid DNA was used to transform E. coli DH10B with selection on ampicillin or cefotaxime. No transformants could be selected on cefotaxime; however, hundreds of colonies were selected on ampicillin. Two different plasmids were identified from different ampicillin-resistant transformants. The first plasmid, labelled pHSN-1, was 53 kb in size, as determined by analysis of its HpaI profile (Figure 1b). PCR and Southern hybridization analysis showed it contained the tet(D) and blaSHV-5 genes (Figure 1b). The presence of blaSHV-5 is responsible for the ceftazidime resistance of the pHSN-1 transformant (Table 1). The second plasmid, pHSN-2, was similarly analysed and found to be 105 kb in size and contained catA1, blaTEM-1 and the class 1 integron containing the two cassettes (Figure 1a). Interestingly,
Isoelectric focusing, and PCR and sequence analysis of antimicrobial resistance genes Isoelectric focusing followed by detection of b-lactamase activity in crude extracts of S. enterica Agona 97-0017 revealed the presence of three b-lactamases with pIs of 8.2, 6.9 and 5.4 (data not shown). PCR was used to detect tetracycline-resistant genes, blaTEM, blaSHV, blaCTX, chloramphenicol resistance genes and class 1 integron cassettes. S. enterica Agona 97-0017 was found to contain the tetracycline-resistance determinant tet(D), the chloramphenicol resistant gene catA1 and two b-lactamase genes including a blaTEM and a blaSHV. In addition, a class 1 integron was identified, which contained a cassette region of 1.7 kb. Sequence analysis identified the b-lactamases as blaTEM-1 and the ESBL blaSHV-5. Sequence analysis of the class 1 integron cassette region identified two novel genes. The first gene displayed 90% identity at the amino acid level to the b-lactamases OXA-2,26 OXA-15,27 and OXA-3228 and was labelled blaOXA-53. The second gene displayed 69% identity at the amino acid level to AAC(60 )-Iq29 and was named aac(6 0 )-I30. Typical attI1 and 59 base elements (59-be) were identified upstream of blaOXA-53 and downstream of aac(6 0 )-I30, respectively. In addition, a 59-be originating in the 30 -end of blaOXA-53 and extending for 69 bp was identified. Thus, the integron contains two cassettes. Although other integrons have been found to contain blaOXA and aac genes in tandem, the BLAST analysis revealed no close homologies from entries in the GenBank database to the blaOXA-53/aac(6 0 )-I30 integron.
Figure 1. HpaI profiles of plasmids isolated from E. coli DH10B transformed with plasmid DNA from S. enterica Agona 97-0017. (a) Plasmid pHSN-2 containing the class 1 integron, catA1 and blaTEM-1, and (b) plasmid pHSN-1 containing tet(D) and blaSHV-5, as determined by PCR and Southern hybridization. (c) Molecular weight marker (One Kilobase Extension Ladder, Invitrogen).
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The clinical isolate S. enterica Agona 97-0017 was originally isolated in 1996 from a 9-month-old female patient in Hospital Sales Neto in Rio de Janeiro, Brazil. This strain was selected for further analysis at the National Microbiology Laboratory, Health _2 Canada, as a representative multidrug-resistant (resistance to > classes of antimicrobials) S. enterica Agona. Initial disc diffusion results showed that the strain was susceptible to ciprofloxacin and trimethoprim/sulfamethoxazole, resistant to chloramphenicol, gentamicin and ampicillin, and intermediate to cefotaxime (data not shown). The cefotaxime results suggested the presence of an ESBL and analysis for the presence of an ESBL was positive using the ceftazidime –ceftazidime/clavulanic acid Etest ESBL strip (Table 1).
The known pIs of blaTEM-1 and blaSHV-5 are 5.4 and 8.2, respectively, so the b-lactamase with a pI of 6.9 was assumed to be blaOXA-53.
Novel integron from Salmonella Agona
the pHSN-2 transformant exhibits a reduced susceptibility to ceftazidime as well when tested by Etest and disc diffusion (Table 1). The pHSN-2 transformant also exhibited resistance to gentamicin, kanamycin and tobramycin (Table 1).
Cloning and analysis of the integron cassette region In order to study the resistance phenotypes associated with the novel integron at the genetic level, it was necessary to isolate the element. The entire cassette region, including the P1 promoter,30 was amplified and cloned into pPCR-Script Cam. Sequence analysis revealed that the integron contained both the weak version of the P1 promoter (TGGACA-[17]-TAAGCT) and the P2 promoter (TTGTTA-[17]-TACAGT).30 The transformant was resistant to a number of b-lactam antimicrobials, notably ceftazidime, and displayed an ESBL phenotype when tested by disc diffusion, similar to the pHSN-2 transformant (Table 1). OXA-15 and OXA-32 are single residue variants of OXA-2 that exhibit an increased resistance to ceftazidime due to D150G
(OXA-15) or L164I (OXA-32) substitutions.26 – 28 OXA-53 has the same residues as OXA-2 at these positions but contains a number of other substitutions compared with OXA-2, notably Q143K, G145D and H155N, which are close to those found in OXA-15 and OXA-32 (Figure 2a). These substitutions are in proximity to two conserved elements of class D b-lactamases (Figure 2a) and thus possibly play a role in the expanded spectrum of OXA-53. We note, however, that OXA-53 is only 90% identical to OXA-2, and the role of other residues in its b-lactam hydrolysis profile is unknown. OXA-53 is also unusual in that it is markedly inhibited by clavulanic acid (Table 1). Among class D enzymes, only OXA-12, OXA-18 and OXA-45 have been found to be inhibited by clavulanic acid similar to the classic class A ESBL phenotype.31 – 33 The transformant harbouring pOA-2 was also resistant to tobramycin and amikacin, but was susceptible to gentamicin, confirming that aac(6 0 )-I30 codes for an aminoglycoside resistance gene (Table 1). The aminoglycoside resistance spectrum is identical to that of strains harbouring aac(6 0 )-Iq.29 An alignment
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Figure 2. Alignments of the proteins coded for by the class 1 integron found in S. enterica Agona 97-0017. (a) OXA-53 aligned with OXA-2 (accession no. P05191) with identical residues indicated by full stops. The single residue differences between OXA-2 and OXA-15 (accession no. AAB05874) and OXA-32 (accession no. AAK58418) are indicated. Conserved regions of class D enzymes are boxed. (b) AAC(60 )-I30 aligned with AAC(60 )-Iq (accession no. AAC25500), AAC(60 )-Ia (accession no. AAA98298), AAC(60 )-Ip (accession no. CAA91010), and AAC(60 )-Ii (accession no. AAB63533). Residues identical to those of AAC(60 )-I30 are boxed and regions conserved amongst these proteins are indicated by asterisks.
M. R. Mulvey et al.
Figure 3. Dendrogram of Salmonella enterica Agona strains used in this study. Hospitals are HMJ, Jesus Children Municipal Hospital; HMSF, Salgado Filho Municipal Hospital; HMSN, Salles Neto Children Municipal Hospital; HUGG, Gaffre´e` Gunille University Hospital; PRONIL, Nilo´polis Children Hospital.
Dissemination of the blaOXA-53/aac(6 0 )-I30 integron The nature of the multidrug resistance found in S. enterica Agona 97-0017 prompted us to examine nine other S. enterica Agona strains collected in Rio de Janeiro in 1996 and forwarded to the National Microbiology Laboratory. Subtyping by PFGE showed the strains could be separated into two clusters with 78% similarity between them (Figure 3). The five strains in one cluster (cluster B, Figure 3) were all isolated from the same hospital and were highly related, showing 86%–100% similarity. The other cluster (cluster A, Figure 3) contained five strains, including S. enterica Agona 97-0017, from four different hospitals, and these strains also exhibited 86%–100% similarity. PCR analysis of the nine additional strains showed they all harboured the catA1 gene and the integron found in S. enterica Agona 97-0017. Seven strains were also positive for a blaTEM gene, a blaSHV gene and tet(D), and hence had identical PCR profiles to S. enterica Agona 97-0017. One strain, 97-0291, was positive for blaSHV but negative for blaTEM and tet(D), and another strain, 97-0484, was negative for blaSHV but positive for blaTEM and tet(D). Thus, although plasmid profiling was not conducted on the additional strains the PCR profiling indicated the likely dissemination of similar plasmids among related strains of S. enterica Agona isolated from different hospitals in Rio de Janeiro in 1996.
Acknowledgements We would like to thank Romeo Hizon for his contribution related to antimicrobial susceptibility testing and Shaun Tyler and the staff of the DNA Core Facility at the NML for generating the sequence information and synthesizing oligonucleotides.
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