Characterization of antimicrobial resistance in Salmonella enterica food and animal isolates from Colombia: identification of a qnrB19-mediated quinolone resistance marker in two novel serovars
Correspondence: Séamus Fanning, Centres for Food Safety & Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: +353 1 716 6082; fax: +353 1 716 6091; e-mail: firstname.lastname@example.org
Ninety-three Salmonella isolates recovered from commercial foods and exotic animals in Colombia were studied. The serotypes, resistance profiles and where applicable the quinolone resistance genes were determined. Salmonella Anatum (n=14), Uganda (19), Braenderup (10) and Newport (10) were the most prevalent serovars, and resistance to tetracycline (18.3%), ampicillin (17.2%) and nalidixic acid (14%) was most common. Nalidixic acid-resistant isolates displayed minimum inhibitory concentrations ranging from 32 to 1024 μg mL−1. A Thr57→Ser substitution in ParC was the most frequent (12 of the 13 isolates). Six isolates possessed an Asp87→Tyr substitution in GyrA. No alterations in GyrA in a further seven nalidixic acid-resistant isolates were observed. Of these, four serovars including two Uganda, one Infantis and a serovar designated 6,7:d:-, all carried qnrB19 genes associated with 2.7 kb plasmids, two of which were completely sequenced. These exhibited 97% (serovar 6,7:d:- isolate) and 100% (serovar Infantis isolate) nucleotide sequence identity with previously identified ColE-like plasmids. This study demonstrates the occurrence of the qnrB19 gene associated with small ColE plasmids hitherto unrecognized in various Salmonella serovars in Colombia. We also report unusual high-level quinolone resistance in the absence of any DNA gyrase mutations in serovars S. Carrau, Muenchen and Uganda.
Salmonellosis is a classic food-borne infection that constitutes a major public health problem. Contaminated food of animal origin including eggs, meat, unpasteurized dairy products, seafood, fruits and vegetables are sources of infections with Salmonella spp. (Mead et al., 1999).
Antimicrobial-resistant salmonellae constitute a health hazard due to the increased risk of therapeutic failure in cases where chemotherapy is indicated. Fluoroquinolones are the drugs of choice to treat invasive, life-threatening salmonellosis. In these zoonotic pathogens, the emergence of fluoroquinolone resistance or reduced susceptibility is particularly challenging (Tollefson et al., 1997; Dimitrov et al., 2007).
Quinolone resistance in Salmonella spp. is principally caused by mutations in the target enzymes, DNA gyrase and topoisomerase IV (Griggs et al., 1996; Piddock et al. 1998; Piddock, 2002; Eaves et al., 2004). Other mechanisms such as increased activity of efflux pumps, decreased permeability due to loss of porins and a variety of plasmid-mediated quinolone resistance (PMQR) mechanisms also contribute to resistance and/or decreased susceptibility, one of the latter being the qnr gene (Martínez-Martínez et al., 1998; Piddock, 2002; Robicsek et al., 2005; Giraud et al., 2006; Strahilevitz et al., 2009).
Rapid dissemination of plasmid-mediated qnr genes has been described in recent years (Robicsek et al., 2006; Cattoir et al., 2007; Hopkins et al., 2007; Minarini et al., 2008; Wu et al., 2008; Cerquetti et al., 2009; Cui et al., 2009; García-Fernández et al., 2009; Gunell et al., 2009). Qnr proteins share common structural properties and belong to a pentapeptide family of proteins. By virtue of their capacity to bind specifically to DNA gyrase, these proteins limit access of the fluoroquinolone drug to its target, thereby providing protection to the bacteria (Tran et al., 2005). Five different qnr genes have been described: qnrA, B, C, D and S with a number of variants exhibiting minor sequence differences (Martínez-Martínez et al., 1998; Hata et al., 2005; Jacoby et al., 2006; Cavaco et al., 2009; Wang et al., 2009). The first qnrB gene described was reported in a Klebsiella pneumoniae isolate from India and was located on a plasmid carrying the blaCTX−M−15-mediated ESL resistance marker (Jacoby et al., 2006). Qnr proteins have been identified in both clinically resistant and susceptible isolates. The minimum inhibitory concentrations (MICs) for nalidixic acid and ciprofloxacin reported in these isolates ranged from twofold to eightfold and 8–32-fold higher, respectively, when compared with the isogenic progenitor isolates (Jacoby et al., 2006; Minarini et al., 2008; Murray et al., 2008; Strahilevitz et al., 2009).
Recently, qnrB determinants were found ubiquitous in commensal microbial communities of healthy children in Peru and Bolivia and were subsequently found to be encoded by small ColE-type plasmids (Pallecchi et al., 2009, 2010).
In this paper, we report on a study of 93 Salmonella isolates recovered from foods and exotic animals in Colombia. Serotyping was used to initially characterize the isolates, and their resistance profiles were determined. A plasmid-mediated qnrB19 marker was detected in four isolates and this gene was completely characterized.
Materials and methods
A collection of 93 Salmonella spp. isolates recovered between 2002 and 2009 from a variety of food products and animals in Colombia was obtained from the University of Cordoba (Colombia). Isolates were streaked on XLD medium (Oxoid, Basingstoke, UK) to check for purity, and were confirmed as Salmonella using a Salmonella latex test (Oxoid).
Drug susceptibility testing
Susceptibilities to 15 drugs were determined by disc diffusion and interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines (2007). The following antimicrobial compounds were used: amoxicillin–clavulanic acid 20/10 μg (AMC), ampicillin 10 μg (AMP), cefpirome 30 μg (CFP), cefpodoxime 10 μg (CPD), ceftiofur 30 μg (CFR), cephalothin 30 μg (KF), chloramphenicol 30 μg (C), ciprofloxacin 5 μg (CIP), gentamicin 10 μg (GM), kanamycin 30 μg (KAN), nalidixic acid 30 μg (NA), neomycin 30 μg (NEO), streptomycin 10 μg (S), trimethoprim/sulfamethoxazole 25 μg (SXT), and tetracycline 30 μg (TE). Discs were purchased from Oxoid. Escherichia coli ATCC® 25922 was included as a control.
MICs for nalidixic acid (Sigma-Aldrich, Ireland) and ciprofloxacin (Sigma-Aldrich) were determined by the broth microdilution method (CLSI, 2007), in the absence and presence of 40 μg mL−1 phe-arg-β-naphthylamide (PAβN) (Sigma–Aldrich).
Nucleic acid purification and DNA sequencing
Genomic DNA extraction, PCR purification and sequencing were performed as described previously (O'Regan et al., 2009). Table 1 provides the details of all primer sequences, annealing temperatures and amplicon sizes. Positive controls for the detection of PMQR genes were included: E. coli Lo qnrA1+, K. pneumoniae B1 qnrB1+, E. coli S7 qnrS1+, E. coli TOP10+pCR2.1W qepA and E. coli 78-01 aac(6′)-Ib-cr+.
Table 1. Target genes, amplification primers and PCR reaction conditions used for characterization of the collection
|TGT CCG AGA TGG CCT GAA GC|
CGT TGA TGC TTC CGT CAG
|55||470||Modified from Carrique-Mas et al. (2008)|
|GAA ATG ACC CGT CGT AAA GG|
TAC AGT CTG CTC ATC AGA AAG
|54||710||O'Regan et al. (2009)|
|ATG AGC GAT ATG GCA GAG CG|
TGA CCG AGT TCG CTT AAC AG
|52||413||Carrique-Mas et al. (2008)|
|GAC CGA GCT GTT CCT TGT GG|
GCG TAA CTG CAT CGG GTT CA
|52||493||Carrique-Mas et al. (2008)|
|GTT GGC GAA AAA ATT GAC AGA A|
|52||500||Wu et al. (2008)|
|TGG ATG GGG ACT CAG GTA CT|
CGG CAC CTG AAA AAT CGC AG
|55||2700||Pallecchi et al. (2010)|
| qnrA1 to qnrA6||QnrAm-F|
|AGA GGA TTT CTC ACG CCA GG|
TGC CAG GCA CAG ATC TTG AC
|54||580||Cattoir et al. (2007)|
| qnrB1 to qnrB6||QnrBm-F|
|GGM ATH GAA ATT CGC CAC TG|
TTT GCY GYY CGC CAG TCG AA
|54||264||Cattoir et al. (2007)|
| qnrS1 to qnrS2||QnrSm-F|
|GCA AGT TCA TTG AAC AGG GT|
TCT AAA CCG TCG AGT TCG GCG
|54||428||Cattoir et al. (2007)|
|TGG TCT ACG CCA TGG ACC TCA|
TGA ATT CGG ACA CCG TCT CCG
|56||1137||M. Galimand, pers. commun.|
|TTG CGA TGC TCT ATG AGT GGC TA|
CTC GAA TGC CTG GCG TGT TT
|55||482||Park et al. (2006)|
| ColE-like plasmid backbone||ColEPB_FW qnrB19ColEPB_RV||CTG ACA CTC AGT TCC GCG A|
CGG CAC CTG AAA AAT CGC AG
|55||900||Pallecchi et al. (2010)|
Nalidixic acid-resistant isolates were assessed for all known PMQR markers using previously published primers (Table 1). Plasmids were purified from nalidixic acid-resistant isolates using the PureYield™ Plasmid Midiprep System (Promega, Madison, WI) and their profiles were determined in a 0.9% agarose gel SeaKem®LE Agarose (Lonza, Wokingham, UK) after electrophoresis in 1 × Tris-HCl (pH 8)–boric acid–EDTA buffer containing 0.1 μg mL−1 ethidium bromide (Sigma-Aldrich). Using a PCR-based method developed previously by Pallecchi et al. (2010), the ColE-like plasmid carrying qnrB19 genetic determinant was amplified and the sequence was determined (Qiagen, Hilden, Germany). Complete amplified plasmid products were subjected to restriction fragment length polymorphism (RFLP) analysis with MboII enzyme (New England Biolabs, Ipswich, MA) to identify any sequence-based polymorphisms.
The complete sequence of these plasmids was determined (Qiagen) and analysed using blast (http://www.ncbi.nlm.nih.gov/BLAST/), clustalw (http://www.ebi.ac.uk/clustalw/) and dnastar (DNAStar Inc., Madison, WI) programs.
Nucleotide sequence accession numbers
Nucleotide sequences determined were deposited in GenBank under accession numbers HM070379 and HM070380.
Results and discussion
Table 2 shows the serovars recovered, along with their source and geographical origin, date of isolation and corresponding susceptibility patterns. In all, 19 serovars were identified, with S. Uganda (n=19), Anatum (n=14), Braenderup (n=10) and Newport (n=10) predominating, followed by serovars Carrau (n=8), Infantis (n=7), Saintpaul (n=5), Muenchen (n=4) and Rubislaw (n=3). Fresno, Javiana and Senftenberg serovars were represented by two isolates each. Single isolates belonging to serovars Adelaide, Bredeney, Derby, Gaminara, Salmonella enterica ssp. enterica 6,7:d:-, Minnesota, and Typhimurium, were also identified in this collection. No Enteritidis serovars were recovered. The most common serovars implicated in human salmonellosis in Colombia are Enteritidis and Typhimurium (Munoz et al., 2006; Wiesner et al., 2006). However, serovars identified in this study have occasionally been implicated in salmonellosis outbreaks worldwide (Lehmacher et al., 1995; Jones et al., 2004; Gupta et al., 2007; Lang, 2008).
Table 2. Characterization of 93 Salmonella enterica isolates from Colombia
|S3||20/10/2004||Cheese||Sincelejo||Anatum||AMP, AMC, KF, TE|
|S20||07/12/2004||Retail chicken||Sincelejo||Infantis||KAN, NEO, NA, S, TE|
|S24||01/02/2005||Sausages||Monteria||Uganda||KAN, NEO, NA, S, TE|
|S36||12/04/2005||Corn and egg mixture||Monteria||Rubislaw||–|
|S38||07/06/2005||Ground meat||Cartagena||Uganda||KAN, NEO, NA, S, TE|
|S39||07/06/2005||Ground meat||Cartagena||Uganda||KAN, NEO, TE|
|S42||22/07/2005||Amazona spp. (parrot)||Monteria||Anatum||–|
|S47||16/08/2005||Ground meat||Cartagena||Uganda||NA, TE|
|S52||18/10/2005||Ground meat||Cartagena||Carrau||NA, TE,|
|S55||18/10/2005||Sausages||Cartagena||Anatum||GM, S, TE|
|S71||10/04/2007||Flour mandioka powder||Monteria||Braenderup||–|
|S72||10/04/2007||Flour mandioka powder||Monteria||Uganda||–|
|S75||16/04/2007||Ground meat||Monteria||6,7:d:-||AMP, NA|
|S87||04/05/2007||Potato and meat||Monteria||Infantis||AMP|
|S91||26/05/2009||Hydrochoerus hydrochaeris (capybara)||Monteria||Fresno||C, KF, TE|
|S92||26/05/2009||Hydrochoerus hydrochaeris||Monteria||Typhimurium||AMC, KF|
|S93||28/05/2009||Trachemys scripta callirostris (hicotea)||Monteria||Javiana||–|
|S95||28/05/2009||Trachemys scripta callirostris||Monteria||Newport||–|
Antimicrobial susceptibility profiles
A summary of the resistance profiles obtained for each isolate against a panel of 15 antimicrobial compounds is shown in Table 3. Forty-six percent (n=40) were resistant to at least one antimicrobial agent. Tetracycline resistance was the most common resistance property encountered (18.3%, n=17), followed by ampicillin resistance (17.2%; n=16), and nalidixic acid resistance (14%; n=13). Multidrug-resistant isolates (defined as resistant to three or more different drug classes) constituted 4.3% of the collection (n=4).
Table 3. Prevalence of antibiotic resistance in the strain collection
|Amoxicillin–clavulanic acid||2.15% (2)|
|Nalidixic acid||14% (13)|
The emergence of quinolone resistance together with reduced ciprofloxacin susceptibility in S. enterica is increasingly observed and constitutes a major concern because infections with such isolates may cause ciprofloxacin treatment failure (Dimitrov et al., 2007). While the frequency of quinolone resistance in Salmonella is growing worldwide, in this study, 14% of the isolates were resistant to nalidixic acid, a figure that could be considered high (Marimón et al., 2004; Stevenson et al., 2007). This corresponded to the data in the SENTRY Antimicrobial Surveillance program, which reported nalidixic acid resistance of 14% in Salmonella spp. isolates from Latin America during the years 1997–2004, a figure more than twofold higher than that recorded in North America (Biedenbach et al., 2006).
In the case of the isolates showing resistance to quinolone-based antimicrobial compounds, an MIC for nalidixic acid of 32 μg mL−1 was recorded for two isolates, 256 μg mL−1 for three, and 1024 μg mL−1 for eight isolates. Reduced susceptibility to ciprofloxacin was noted for all 13 isolates (ranging from 0.5 to 1 μg mL−1). A summary of the MIC data is presented in Table 4.
Table 4. MIC values and PCR analysis of target genes associated with resistance in 13 Salmonella isolates displaying (fluoro)quinolone resistance
|S20/chicken/Infantis||qnrB19||32 – 4 (8 ×)||1||WT||WT||Thr57→Ser||WT|
|S24/sausages/Uganda||qnrB19||32 – 4 (8 ×)||1||WT||WT||Thr57→Ser||WT|
|S37/potato/Muenchen||–||1024 – 128 (8 ×)||1||WT||WT||Thr57→Ser||WT|
|S38/ground meat/Uganda||qnrB19||256 – 64 (4 ×)||1||WT||WT||Thr57→Ser||WT|
|S44/ground meat/Anatum||–||1024 – 64 (16 ×)||0.5||Asp87→Tyr||WT||Thr57→Ser||WT|
|S45/meat/Carrau||–||256 – 64 (2 ×)||0.5||Asp87→Tyr||WT||Thr57→Ser||WT|
|S46/cheese/Carrau||–||1024 – 512 (2 ×)||0.5||Asp87→Tyr||WT||Thr57→Ser||Asn446→Pro|
|S47/ground meat/Uganda||–||1024 – 256 (4 ×)||0.5||WT||WT||Gly25→Ala|
|S51/cheese/Carrau||–||1024 – 512 (2 ×)||0.5||Asp87→Tyr||WT||Thr57→Ser||WT|
|S52/ground meat/Carrau||–||1024 – 512 (2 ×)||1||WT||WT||Thr57→Ser||Arg508→Lys|
|S53/ham/Anatum||–||1024 – 512 (2 ×)||0.5||Asp87→Tyr||WT||Thr57→Ser||Arg508→Lys|
|S64/cheese/Muenchen||–||1024 – 512 (2 ×)||0.5||Asp87→Tyr||WT||Thr57→Ser||WT|
|S75/ground meat/6,7:d:-,||qnrB19||256 – 128 (2 ×)||1||WT||WT||WT||WT|
A 2–16-fold decrease in the MIC of nalidixic acid was observed In the presence of PAβN, a known efflux pump inhibitor (Table 4) with six isolates showing a 4–16-fold decrease. These results indicate that efflux pump activity may be contributing to the resistant phenotype and will be investigated in a separate follow-up study.
Target gene mutations
Target gene mutations associated with resistance to fluoroquinolones/quinolones (F)Q are shown in Table 4. One of the isolates did not possess any target gene mutations. Others possessed up to three mutations in the corresponding target genes. Six of 13 nalidixic acid-resistant isolates had mutations in the QRDR region of gyrA; in all these cases the Asp87→Tyr substitution was noted. No amino acid sequence changes were identified in GyrB. Substitutions in ParC (Thr57→Ser) were noted in 12 isolates. One had a Gly25→Ala along with a second substitution within ParC (isolate S47, Table 4). Two different ParE mutations were identified: Asn446→Pro in one isolate (S46) and Arg508→Lys in another two isolates (S52 and S53, Table 4).
High-level resistance to nalidixic acid and decreased susceptibility to ciprofloxacin was observed in isolates S44, S45, S46, S51, S53 and S64, which could be attributed to the single substitution in the GyrA previously found to correlate with this phenotype (Walker et al., 2001; Eaves et al., 2002; Ling et al., 2003; Stevenson et al., 2007). In isolates S20, S24, S38 and S75, nalidixic acid resistance could be attributed to the presence of PMQR. Characteristically, nalidixic acid MICs in these latter isolates were lower (ranging from 32 to 256 μg mL−1) compared with isolates with the more common gyrA mutation. However, three remaining isolates of serovars Muenchen (denoted as S37), Uganda (S47) and Carrau (S52) did not possess GyrA substitutions, but were highly resistant to nalidixic acid (MIC=1.024 μg mL−1) and displayed reduced susceptibility to ciprofloxacin (MIC=0.5–1 μg mL−1). All three possessed the Thr57→Ser ParC substitution. Salmonella Uganda (S47) also contained a second ParC amino acid change (Gly25→Ala), and the Carrau isolate (S52) had an additional Arg508→Lys substitution in ParE. Because these isolates possessed different mutations, it was difficult to conclude as to which mechanism was primarily responsible for the phenotype observed. Contribution of increased efflux activity is likely in the S. Muenchen and Uganda isolates as demonstrated by the MIC assay in the presence of PAβN. Nonetheless, MICs decreased to 128 and 256 μg mL−1 in these two isolates, respectively, values that are indicative of clinical resistance, strongly suggesting the presence of (an) additional undefined mechanism(s).
Some reports suggest that the distribution of specific substitutions within target genes might differ depending on the serovar. Furthermore, the frequency with which these mutations are observed may reflect the impact of exposure to different fluoroquinolone drugs (Giraud et al., 1999; Levy et al., 2004). Nonetheless, mutation patterns in the isolates studied could not be correlated with specific serovars. The spectrum of mutations observed was narrow and only one polymorphism occurred in gyrA, suggesting that conditions under which the mutations developed might have been a common factor leading to this particular alteration.
The lack of gyrA mutations in some isolates together with the presence of parC mutations in six other isolates is a unique finding. Although the Thr57→Ser substitution in ParC has been reported previously in Salmonella, it is detected less frequently compared with the more common gyrA mutations and typically occurs concomitantly with double gyrA mutations (Piddock et al. 1998; Baucheron et al., 2005; Hopkins et al., 2005). The Thr57→Ser mutation in parC was first reported by Ling et al. (2003) in Salmonella isolates with a wild-type DNA gyrase and others possessing single gyrA mutations, wherein the first were susceptible to ciprofloxacin (MIC=0.06 μg mL−1), and the latter demonstrated a twofold increased resistance. More recently, Baucheron et al. (2005) reported that the Thr→57Ser ParC substitution was not involved in quinolone resistance in their isolates. Also, Cui et al. (2009) reported an identical ParC substitution in a ciprofloxacin-resistant S. Rissen isolate that did not carry any other target gene mutation, qnr alleles nor an aac-(6′)-Ib-cr gene. In addition, the same polymorphism was recently encountered in a number of non-Typhimurium isolates and the resistant phenotype could not be linked with this alteration because susceptible isolates harboured identical mutations (Gunell et al., 2009). Thus, we also sequenced the parC gene of 10 randomly selected quinolone-susceptible isolates from this collection representing five serotypes. Thr→57Ser substitution was identified in nine of 10 of these isolates (data not shown), supporting the view that this is a common polymorphism in serotypes other than Typhimurium. In view of current knowledge regarding quinolone resistance mechanisms, it is unclear whether secondary target mutations alone can lead to the development of high-level quinolone resistance (Ling et al., 2003; Baucheron et al., 2005; Cui et al., 2009; Gunell et al., 2009).
Detection of PMQR
PCR analysis of the fluoroquinolone-resistant isolates did not detect aac(6′)-Ib-cr, qepA, qnrA nor qnrS genes. Four isolates were positive for qnrB (Table 4): one Infantis (S20), two Uganda isolates (S24, S38) and one serovar 6,7:d:- isolate (S75). The MICs of nalidixic acid in these isolates varied from 32 to 256 μg mL−1. DNA sequencing revealed the presence of the qnrB19 allele in all cases. Multiple plasmids were present in nine isolates (data not shown) while four other isolates (denoted as S37, S45, S47 and S51) lacked detectable plasmids. In the plasmid-positive qnrB19 isolates S20, S24, S38 and S75, several other low-molecular-weight plasmids ranging in size between 1 and 3 kb were also noted (data not shown). When analysed by PCR designed to amplify ColE-like plasmids, amplicons of 2.7 kb were recovered. Among these, two distinct MboII RFLP profiles were observed, which were identical for three isolates (S20, S24, and S38), and different for isolate S75 (data not shown). The plasmids from isolates S20 and S75 were purified and completely sequenced.
Homology searches revealed that the plasmid (designated pMK100) found in S. Infantis (S20) exhibited 100% homology with qnrB19-carrying plasmids including pSGI15, a small ColE plasmid identified recently in S. enterica serovar Typhimurium isolated in Germany (Hammerl et al., 2010), and a qnrB19-containing plasmid pPAB19 from an S. Infantis clinical isolate recovered in Argentina (GenBank accession number GQ412195). The plasmid purified from isolate S75 (designated pMK101) was found to be 97% similar to these latter plasmids. The dissimilarity noted was mapped to an insertion located between nucleotide positions 896 and 957. Remarkably, the latter DNA sequence was identical to one found in a pBC633 from a K. pneumoniae strain KN633 (accession number EU176012), a urinary isolate from Colombia displaying carbapenem resistance and reported in 2005. This plasmid of approximately 15.5 kb carried a blaKPC−2 gene encoding a class A carbapenemase (Villegas et al., 2006). The additional DNA sequence contained in the plasmid from the isolate S75 was located between the qnrB19 gene and orf2, and was found to be homologous with a region of pBC633. Furthermore, nucleotide sequence similarity was observed in the region upstream of the inserted fragment, possibly facilitating the incorporation of the new DNA fragment. The fact that pBC633 was found only in Colombia indicates that the homology found here may not be coincidental. It is interesting to speculate that pMK101 (the plasmid from isolate S75) is chimeric and may have emerged as a result of a recombination event that led to the horizontal acquisition of a fragment from another plasmid containing blaKPC−2. The process is likely to have occurred in a bacterium simultaneously hosting a plasmid similar to or identical to pBC633, as well as a small ColE-like plasmid such as pMK100. While blaKPC−2 genes are frequent in K. pneumoniae and only sporadic in other Enterobacteriaceae, there are insufficient data to conclude what species was the primary host of the new plasmid structure (Villegas et al., 2006; Pournaras et al., 2009). In addition, it is noteworthy that pBC633 containing a blaKPC−2 gene was found on a transposon Tn4401 with multiple insertion sequence (IS) elements that have likely contributed to its emergence (Naas et al., 2008). Of particular concern is the possibility of the emergence of chimeric plasmids carrying both qnr genes and blaKPC−2 that could compromise the clinical value of fluoroquinolones and virtually all β-lactams. In view of this, monitoring of phenotypic resistance as well as associated mechanisms and mobility is essential. Furthermore, the occurrence of both blaKPC−2 and qnr in Colombia and their associated plasmids is likely to be under-reported as a result of poor surveillance as well as diagnostic challenges associated with the low-level resistance conferred (Villegas et al., 2006). The present study documented the emergence of a plasmid carrying a sequence previously identified in another plasmid and suggests that reservoirs of both types of plasmids may exist in Colombia.
In Colombia, epidemiological data relating to PMQR is limited. A single case reporting PMQR in Colombia described the qnrB19 gene in E. coli isolates recovered from blood cultures of a hospital patient in Monteria (Cattoir et al., 2008). The gene was linked with ISEcp1-like insertion element responsible for its mobilization and was carried by a novel transposon designated Tn2012 identified on pR4525 (Cattoir et al., 2008). No linkage of qnrB19 with transposon or integron structures was observed in our isolates (data not shown). A high prevalence of qnrB determinants was reported recently in commensal microbial communities cultured from healthy children in Peru and Bolivia (Pallecchi et al., 2009). In a follow-up study, the involvement of ColE-type plasmids and their role in dissemination in these two countries was described (Palecchi et al., 2010). The most prevalent plasmid, designated pECY6-7, was investigated in detail, and was found to be identical to the plasmid characterized by Hammerl et al. (2010). Both plasmids are indistinguishable from those characterized in the S. Infantis isolate (denoted as S20).
These data extend our understanding of the molecular epidemiology of the qnrB19 determinant. In this study, the marker was identified for the first time in Salmonella spp. in Colombia. The fact that the isolates include different serovars, and that they were recovered in different areas of the country from a variety of food samples and over the years (2002–2009), suggests that the reservoir may not be restricted to a specific ecological niche. Further epidemiological studies are required to determine the full extent of the dissemination of PMQR in Colombia and its implications for public health.
The authors acknowledge financial support from the Research Stimulus Fund of the Department of Agriculture, Fisheries and Food of Ireland (RSF) (06/TNI-UCD10) and COST (ATENS) grant COST-STSM-BM0701-05056. Bacterial isolates E. coli Lo qnrA1+, K. pneumoniae B1 qnrB1+ and E. coli S7 qnrS1+ were a kind gift from Professor Patrice Nordmann, E. coli TOP10+pCR2.1WqepA was kindly provided by Dr Marc Galimand and E. coli 78-01 aac(6′)-Ib-cr+ by Professor Johann Pitout.