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Research letters
might have occurred in Enterobacteriaceae originating from an
animal host.
The presence of the qnrB2 gene in animal isolates and zoonotic pathogens opens the possibility that genetic exchange and
plasmid acquisition of the qnrB2 gene could occur in the faecal
flora of the animals. Interestingly, p137.25 belongs to the IncN
plasmid family that is able to replicate in different enterobacterial strains, but also seems prevalent in faecal flora from animals.
In fact, a study performed on a large collection of E. coli from
the USA demonstrated that the prevalence of IncN plasmids is
high in avian E. coli (10% – 16%) but negative in E. coli from
faeces of healthy humans.15 This evidence supports the hypothesis that the Salmonella 137.25 strain acquired the qnrB2 gene
on an IncN plasmid circulating in avian bacterial flora. This
strain could cause infections in humans through the food chain
and the resistance plasmid contributes to the dissemination of
the qnrB2 gene in other Enterobacteriaceae.
10. Espedido BA, Partridge SR, Iredell JR. blaIMP-4 in different
genetic contexts in Enterobacteriaceae isolates from Australia.
Antimicrob Agents Chemother 2008; 52: 2984–7.
11. Chen YT, Liao TL, Liu YM et al. Mobilization of qnrB2
and ISCR1 in plasmids. Antimicrob Agents Chemother 2009; 53:
1235–7.
12. Gutierrez B, Herrera-Leon S, Escudero JA et al. Novel genetic
environment of qnrB2 associated with TEM-1 and SHV-12 on pB1004,
an IncHI2 plasmid, in Salmonella Bredeney BB1047 from Spain.
J Antimicrob Chemother 2009; 64: 1334–6.
13. Pomba C, da Fonseca JD, Baptista BC et al. Detection of the
pandemic
O25-ST131
human
virulent
Escherichia
coli
CTX-M-15-producing clone harbouring the qnrB2 and aac(6 0 )-Ib-cr
genes in a dog. Antimicrob Agents Chemother 2009; 53: 327–8.
14. Carattoli A. Resistance plasmid families in Enterobacteriaceae.
Antimicrob Agents Chemother 2009; 53: 2227–38.
15. Johnson TJ, Wannemuehler YM, Johnson SJ et al. Plasmid
replicon typing of commensal and pathogenic Escherichia coli isolates.
Appl Environ Microbiol 2007; 73: 1976–83.
Funding
This work has been partly funded by Med-Vet-Net,
Workpackages 21 and 29. Med-Vet-Net is an EU-funded
Network of Excellence.
Journal of Antimicrobial Chemotherapy
doi:10.1093/jac/dkp340
Advance Access publication 22 September 2009
Novel genetic environment of qnrB2 associated with
TEM-1 and SHV-12 on pB1004, an IncHI2 plasmid,
in Salmonella Bredeney BB1047 from Spain
Transparency declarations
None to declare.
References
1. Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence
of plasmid-mediated quinolone resistance. Lancet Infect Dis 2006; 6:
629–40.
2. Cerquetti M, Garcı́a-Fernández A, Giufrè M et al. First report of
plasmid-mediated quinolone resistance determinant qnrS1 in an
Escherichia coli strain of animal origin in Italy. Antimicrob Agents
Chemother 2009; 53: 3112– 4.
3. Ma J, Zeng Z, Chen Z et al. High prevalence of plasmid-mediated
quinolone resistance determinants qnr, aac(6 0 )-Ib-cr, and qepA among
ceftiofur-resistant Enterobacteriaceae isolates from companion and
food-producing animals. Antimicrob Agents Chemother 2009; 53:
519–24.
4. Yue L, Jiang HX, Liao XP et al. Prevalence of plasmid-mediated
quinolone resistance qnr genes in poultry and swine clinical isolates of
Escherichia coli. Vet Microbiol 2008; 132: 414– 20.
5. Liu JH, Deng YT, Zeng ZL et al. Coprevalence of plasmidmediated quinolone resistance determinants QepA, Qnr, and
AAC(60 )-Ib-cr among 16S rRNA methylase RmtB-producing
Escherichia coli isolates from pigs. Antimicrob Agents Chemother
2008; 52: 2992–3.
6. Gay K, Robicsek A, Strahilevitz J et al. Plasmid-mediated quinolone resistance in non-Typhi serotypes of Salmonella enterica. Clin
Infect Dis 2006; 43: 297– 304.
7. Fortini D, Garcı́a-Fernández A, Veldman K et al. Novel genetic
environment of plasmid-mediated quinolone resistance gene qnrB2 in
Salmonella Bredeney from poultry. J Antimicrob Chemother 2009; 64:
1332–4.
8. Garcı́a A, Navarro F, Miró E et al. Characterization of the highly variable region surrounding the blaCTX-M-9 gene in non-related Escherichia coli
from Barcelona. J Antimicrob Chemother 2005; 56: 819–26.
9. Wu JJ, Ko WC, Chiou CS et al. Emergence of Qnr determinants
in human Salmonella isolates in Taiwan. J Antimicrob Chemother
2008; 62: 1269–72.
Belén Gutierrez1,2, Silvia Herrera-Leon3,
José Antonio Escudero1,2, Laura Hidalgo1,2,
Rubén Gonzalez-Sanz3, Margarita Arroyo3,
Álvaro San Millan1,2, Marı́a Aurora Echeita3 and
Bruno Gonzalez-Zorn1,2*
1
Departamento de Sanidad Animal, Facultad de Veterinaria,
Universidad Complutense de Madrid, Spain; 2Centro de
Vigilancia Sanitaria Veterinaria, Universidad Complutense
de Madrid, Spain; 3Laboratorio de Referencia de
Salmonella, Centro Nacional de Microbiologı́a, Instituto de
Salud Carlos III, Madrid, Spain
Keywords: ESBLs, fluoroquinolones, pEC-IMPQ
*Corresponding author. Departamento de Sanidad Animal,
Facultad de Veterinaria, Universidad Complutense de Madrid,
28040 Madrid, Spain. Tel: þ34-91-394-37-07; Fax: þ34-91394-39-08; E-mail: [email protected]
Sir,
Resistance to fluoroquinolones through plasmid-mediated qnr
genes, especially when associated with resistance to cephalosporins by extended-spectrum b-lactamases (ESBLs), is an
emerging phenomenon in enterobacteria. This is worrisome in
the case of Salmonella, as these are two of the major antimicrobials used in invasive salmonellosis.1 We report here the presence of a qnrB2 gene in a Salmonella Bredeney clinical isolate,
BB1047, from Spain. The gene was borne by a self-transferable
IncHI2 plasmid, pB1004, associated with blaTEM-1 and
blaSHV-12. The genetic environment of qnrB2 revealed a
common evolutionary origin with plasmid pEC-IMPQ recently
described in Enterobacter cloacae clinical isolates from Taiwan.
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Research letters
In 2005, a 76-year-old woman in an intensive care unit in South
Spain suffered from insidious wound infection. Microbial analysis showed that Salmonella Bredeney, BB1047, was the causative agent. Complete antimicrobial profile revealed that the
bacterium possessed an unusual multidrug resistance profile
[Table S1, available as Supplementary data at JAC Online
(http://jac.oxfordjournals.org/)]. In order to characterize the
genetic basis of the resistance profile, conjugation experiments
were performed. Resistance to b-lactams, aminoglycosides,
chloramphenicol, tetracycline and fluoroquinolones could be
transferred en bloc into Escherichia coli K802N (res –, gyrA,
NalR) at a frequency of 110 – 5 per donor cfu, giving rise to
transconjugant BB1048 (Table S1). Using the S1-PFGE method,
the size of the plasmid in BB1047 and BB1048 was estimated to
be 315 kbp, and was named pB1004 (data not shown).
Hybridization with b-lactamase probes (OXA, CMY, CTX,
TEM and SHV) and sequencing revealed that the genes encoding TEM-1 and SHV-12 were located in pB1004. In order to
characterize pB1004, the plasmid was extracted, digested with
HindIII, ligated into pUC19, transformed into E. coli and
plated on brain heart infusion agar plates containing ampicillin
(50 mg/L). Two different clones were obtained, bearing a
7613 bp and a 4489 bp fragment that were completely sequenced
in both strands by genome walking (GenBank accession
numbers EU643617 and FJ973574). The 4489 bp fragment
encoded the trhA and trhL genes, as well as most of the putative
replication origin of the plasmid. It was 100% identical to the
IncHI2 plasmids pK29 from Klebsiella pneumoniae (GenBank
accession number EF382672), R478 from Serratia marcescens
(GenBank accession number BX664015) and the recently
described plasmids pEC-IMP and pCE-IMPQ in E. cloacae
from Taiwan.2 When compared with the rest of the sequences
present in the databases, the fragment differed by 26 nucleotides
from other IncHI2 plasmids, like plasmid pAPEC-O1-R from
E. coli (GenBank accession number DQ517526). Thus, pB1004
can be assigned to the R478 subfamily of the IncHI2 plasmids
described to date.
Sequence analysis of the 7613 bp revealed the presence of
the qnrB2 gene, the structural gene of the QnrB2 protein.3 To
assess whether qnrB2 was responsible for quinolone resistance
in this strain (Table S1), the gene was amplified together with
the putative promoter and terminator regions with primers
qnrB2F (50 -CGACCGGGAAAATTGACATG-30 ) and qnrB2R
(50 -CAGGGATGGATTTCAGAACC-30 ), and cloned into
pCR2.1. The resulting transformant, BB1049, showed reduced
susceptibility to quinolones and fluoroquinolones (Table S1)
demonstrating that qnrB2 contributes to the phenotype of
BB1047 against these antimicrobials.
The qnrB2 gene was described for the first time in
Citrobacter koseri (GenBank accession number DQ351242),
and was detected thereafter in Proteus mirabilis (GenBank
accession number EF488762), E. cloacae (GenBank accession
number EU131184), Citrobacter freundii (GenBank accession
numbers AB281054 and FJ232918), K. pneumoniae (GenBank
accession numbers AJ609296 and FJ943244) and Salmonella
enterica serovars Enteritidis,4 Keurmassar5 and Mbandaka. The
Figure 1. Schematic representation of the genetic environments of the qnrB2 gene described to date. Grey shading denotes 99% nucleotide identity. Black
lines beneath Salmonella Bredeney indicate the regions that have been amplified by overlapping PCR and sequenced using the nucleotide sequence of
pEC-IMPQ for the design of appropriate primers. This figure appears in colour in the online version of JAC and in black and white in the print version
of JAC.
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Research letters
genetic environment of qnrB2 has been described in the
Salmonella Enteritidis and Keurmassar serovars, as well as in
the Klebsiella strains and partially in C. koseri, being highly
similar in all bacteria described to date (Figure 1). In pB1004,
immediately upstream of qnrB2, an IS26 is followed by two
genes homologous to genes located in the chromosome of the
marine bacterium Marinobacter aquaeolei VT8 (GenBank
accession number CP000514). The intM gene encodes IntM, a
513 amino acid protein with 68.3% identity with Maqu_0026,
encoding the catalytic domain of the IS21 transposase.
Downstream of intM, istB codes for IstB, an ATP-binding
protein with 83.4% identity with Maqu_0025 of M. aquaeolei
involved in the transposition of IS21-like elements. The two
genes are located in the same order in the chromosome of
M. aquaeolei VT8. Interestingly, both genes are present in the
recently described Taiwanese InHI2 plasmids pEC-IMP and
pEC-IMPQ, the latter also bearing the qnrB2 gene. Further
analysis by PCR and sequencing of the genetic environment of
qnrB2 revealed the existence of an 8939 bp deletion including
an ISCR1 element and a class I integron with the blaIMP-8
metallo-b-lactamase gene.
Here we report the identification of a qnr gene in Salmonella
in Spain. Previously, qnrB2 has been identified in Enterobacter
spp. and qnrA has been detected in E. coli, E. cloacae and
K. pneumoniae, whereas the qnrS gene has been shown to be
present in a K. pneumoniae clinical isolate in this country. In
other countries like France, the UK, Germany, Israel, Australia,
the USA, Taiwan, the Netherlands and Senegal, the qnrB gene
is largely present.6 In the latter four, Salmonella spp. was the
host bacterium of the qnrB2 gene. In Taiwan, the gene was
present in the serovar Enteritidis,4 and possessed a similar
genetic environment to that described previously for Salmonella
Keurmassar.5 Interestingly, the single qnrB2-bearing Salmonella
originating from a Dutch broiler chicken also belongs to serovar
Bredeney (isolate 137.25)6 and was suspected to be potentially
linked to the Bredeney isolate from Spain. However, the genetic
environment flanking the qnrB2 gene and the plasmid incompatibility groups are completely different for the two Bredeney isolates from the Netherlands and Spain (IncN-p137.25 and
IncHI2-pB1004, respectively), suggesting independent events of
acquisition of qnrB2-carrying plasmids in these isolates.7
In contrast, the genetic environment of this qnrB2, as well as
the partial sequence of the plasmid backbone, reveals a striking
common evolutionary origin of pB1004 with pEC-IMPQ. This
is further supported by the plasmid size of a Taiwanese plasmid
that has been shown to be 324 kbp,2 as compared with the
315 kbp from pB1004. It is tempting to speculate that the ISCR1
element has been responsible for the deletion of the 8939 bp in
pB1004 through rolling-circle replication. However, detailed
analysis of the junctions reveals that the deletion occurred
upstream of the replication origin of the ISCR1 element.
Further, a perfect IRL (‘left inverted repeat’) structure in the
IS26 element downstream of qnrB2 is evident in pB1004, indicating that insertion of IS26 occurred after deletion of the
8939 bp fragment. These results confirm emergence in Spain of
pB1004, an IncHI2 plasmid related to pEC-IMPQ, that associates the qnrB2 gene with SHV-12 and TEM-1.
Funding
This work was supported by WP29 of the Network of
Excellence FOOD-CT-506122 MED-VET-NET and the
MICINN (GEN2006-27767-E/PAT). J. A. E., A. S. M. and
L. H. acknowledge the Universidad Complutense de Madrid, the
Spanish Ministry of Education and Science and the Comunidad
de Madrid for their respective fellowships.
Transparency declarations
None to declare.
Supplementary data
Table S1 is available as Supplementary data at JAC Online
(http://jac.oxfordjournals.org/).
References
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resistance in Salmonella enterica, United Kingdom. Emerg Infect Dis
2008; 14: 340–2.
2. Chen YT, Liao TL, Liu YM et al. Mobilization of qnrB2 and ISCR1
in plasmids. Antimicrob Agents Chemother 2009; 53: 1235–7.
3. Jacoby GA, Walsh KE, Mills DM et al. qnrB, another plasmidmediated gene for quinolone resistance. Antimicrob Agents Chemother
2006; 50: 1178–82.
4. Wu JJ, Ko WC, Chiou CS et al. Emergence of Qnr determinants
in human Salmonella isolates in Taiwan. J Antimicrob Chemother
2008; 62: 1269–72.
5. Garnier F, Raked N, Gassama A et al. Genetic environment of
quinolone resistance gene qnrB2 in a complex sul1-type integron in the
newly described Salmonella enterica serovar Keurmassar. Antimicrob
Agents Chemother 2006; 50: 3200–2.
6. Veldman K, van Pelt W, Mevius D. First report of qnr genes
in Salmonella in The Netherlands. J Antimicrob Chemother 2008; 61:
452–3.
7. Fortini D, Garcı́a-Fernández A, Veldman K et al. Novel genetic
environment of plasmid-mediated quinolone resistance gene qnrB2 in
Salmonella Bredeney from poultry. J Antimicrob Chemother 2009; 64:
1332–4.
Journal of Antimicrobial Chemotherapy
doi:10.1093/jac/dkp375
Advance Access publication 21 October 2009
In vitro activity of the new quinolone derivative RD-3
against clinical isolates of Mycoplasma pneumoniae
and Mycoplasma hominis
Shilpakala Sainath Rao* and Malathi Raghunathan
Department of Genetics, Dr ALMPGIBMS, University
of Madras, Taramani Campus, Chennai 600113, India
Keywords: MICs, MBCs, fluoroquinolones
Acknowledgements
N. Montero is acknowledged for excellent technical assistance.
*Corresponding author. Tel: þ91-044-24925317;
Fax: þ91-044-24926709; E-mail: [email protected]
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