Short Communication J Mol Microbiol Biotechnol 2014;24:130–134 DOI: 10.1159/000362278
Published online: May 22, 2014
Dissemination of Bacterial Fluoroquinolone Resistance in Two Multidrug-Resistant Enterobacteriaceae Carina M. Jung Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, Miss., USA
Key Words Horizontal gene transfer · Conjugation · Fluoroquinolone
Abstract Bacterial resistance to antimicrobials has become one of the greatest challenges for clinical microbiologists and healthcare practitioners worldwide. Acquisition of resistance genes has proven to be difficult to characterize and is largely uncontrollable in the environment. Here we sought to characterize conjugal horizontal gene transfer of plasmid-encoded fluoroquinolone resistance genes from two strains of Enterobacteriaceae, one clinical and one from a municipal wastewater treatment plant environment. Conjugation was dissimilar between the two strains. Escherichia coli strain LR09, containing a plasmid with the aac(6′)-Ib-cr fluoroquinolone resistance gene, did not conjugate with any of the 15 strains tested, while Enterobacter aerogenes strain YS11 conjugated with two strains of E. coli. The resultant transconjugants were also dissimilar in their stability and potential persistence. The observations presented herein exemplify the difficulties in understanding and controlling the spread of antimicrobial resistance. Thus, it may be prudent to address drug disposal and destruction, incorporating a life-cycle assessment plan ‘from cradle to grave’, treating antimicrobials as chemical or environmental contaminants. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1464–1801/14/0242–0130$39.50/0 E-Mail [email protected] www.karger.com/mmb
Bacterial resistance to fluoroquinolone (FQ) antimicrobials has become an increasingly serious issue over the past 10 years. Chromosomal point mutations in the quinolone resistance-determining region of DNA gyrase/ topoisomerase IV responsible for blocking the binding of FQs were first recognized in the 1980s [Dalhoff, 2012; Jacoby, 2005; Schmitz et al., 1998]. Efflux pump activity was then noted as a mechanism to decrease the effective concentration of the drug in the cell [Baucheron et al., 2004; Hopkins et al., 2005]. These chromosomal mutations, although heritable, are not generally considered to be disseminated to a larger population. However, later observations led to the discovery of plasmid-derived, transferable resistance mechanisms. The qnr system of pentapeptide repeat molecules serves to mimic the DNA gyrase/topoisomerase IV and reduces the effective intracellular concentration of the drug [Hata et al., 2005; Hegde et al., 2005; Jacoby et al., 2006; Martínez-Martínez et al., 1998]. The aac(6′)-Ib-cr gene is responsible for direct modification, through N-acetylation of the piperazine moiety of many FQs [Domagala, 1994; Jung et al., 2009], while QepA, a plasmid-associated efflux pump, clears FQs from the cell [Park et al., 2009; Yamane et al., 2007]. Views, opinions, and/or findings contained in this paper are those of the authors and should not be construed as an official Department of the Army position or decision unless so designated by other official documentation.
Carina M. Jung CEERD-EP-P US Army Engineer Research and Development Center 3909 Halls Ferry Road, Vicksburg, MS 39180 (USA) E-Mail Carina.M.Jung @ usace.army.mil
Table 1. Bacterial strains and selective media used in conjugation
Many characterized plasmids carrying FQ resistance genes have numerous transposable elements and similarities in various regions to other plasmids [Park et al., 2009; Rodriquez-Martinez et al., 2011]. It can be assumed that these plasmids are mobile and have gone through recombination events in their environment. We were interested in determining the prevalence of horizontal gene transfer (HGT) from bacterial strains harboring FQ-resistant plasmids to other bacteria of various lineages and conducted mating tests with two multidrug FQ-resistant Enterobacteriaceae. Escherichia coli strain LR09 [Jung et al., 2009], a wastewater treatment plant isolate from Little Rock, Arkansas, and Enterobacter aerogenes strain YS11 [Park et al., 2009], which is a clinical isolate from South Korea, were employed as plasmid (IncF) donors. Strain LR09 is resistant to FQs, β-lactams, aminoglycosides, and chloramphenicol. FQ resistance is conferred through mutations in the gyrA and parC genes of the quinolone resistance-determining region, active efflux mechanism(s), and a plasmid-encoded aac(6′)-Ib-cr gene [Jung et al.,
2009]. Strain YS11 contains a plasmid with qnrS1 and qepA FQ resistance genes as well as the genes for β-lactam and aminoglycoside resistance, blaTEM-1 and blaLAP-1, and rmtB, respectively. Mating recipients were first screened for sensitivity to norfloxacin at 1 ng/μl, then sodium azide (NaN3)-resistant mutants were generated as a means to counterselect against donors. A 25-μl drop of 100 μg/μl NaN3 was placed in the center of a spread plate of each culture and colonies growing closest to the NaN3 spot were passaged three times in Luria-Bertani (LB) broth + 100 ng/μl NaN3. A single NaN3-resistant mutant was isolated and used as a conjugation recipient strain for mating against FQ-resistant donors. E. coli strain J53 was already resistant to NaN3 and was excluded from mutant generation. Further, as a means to combat background NaN3-resistant donors, selective and differential media were used to isolate or differentiate transconjugants from donor strains (table 1). All media contained 100 ng/μl NaN3 and either 1 ng/μl or 5 ng/μl norfloxacin. Conjugal mating was established by growing overnight cultures of recipients in LB + 100 ng/μl NaN3 and donors in LB + 5 ng/μl norfloxacin. Cultures were pelleted and washed in sterile saline (0.85% NaCl). Donors were added to 5 ml LB at a final OD600 of 0.02, and recipients were added at a final OD600 of 0.2. Liquid mating in LB commenced for 24 h at 150 rpm on a platform shaker before the conjugation mixture was distributed to the appropriate selective plates (table 1). Various concentrations of donor:recipient (1:1, 1:2, 1:5, 1:10), addition of 1 ng/μl norfloxacin to the LB mating mixture, and filter mating were compared to the method listed above, but no measurable advantage was observed (data not shown). Differing total cell concentrations were not tested, although there is evidence that lower cell concentrations may increase mating efficiencies for IncF plasmids [Reisner et al., 2012]. Only two conjugal pairs, both recipients being E. coli strains, were observed with E. aerogenes strain YS11 (QepA+). The efficiency of conjugation for both YS11:J53 and YS11:S17 mating pairs were on the order of 10–8. No conjugation was observed with E. coli strain LR09. This may be due in part to the level of norfloxacin used in the screening, since aac(6′)Ib-cr potentially was the only FQ resistance gene being transferred. It is thought that aac(6′)-Ib-cr confers, at most, an around 10-fold increase in minimal inhibitory concentration (MIC) [Frasson et al., 2011; Xu et al., 2007]. If a recipient strain was extremely sensitive to norfloxacin, 1 ng/μl would have been too high of a screening level. Lowering the concentration of norfloxa-
Dissemination of Bacterial Fluoroquinolone Resistance
J Mol Microbiol Biotechnol 2014;24:130–134 DOI: 10.1159/000362278
trials Organism Donor Enterobacter aerogenes YS11
Recipients Bacillus cereus NCTR-466 Escherichia coli ATCC 47004 Escherichia coli strain J53 Escherichia coli strain S17 Escherichia coli ATCC 25922 Klebsiella pneumoniae ATCC 13883 Proteus mirabilis ATCC 7002 Pseudomonas fluorescens IC Pseudomonas aeruginosa ATCC PA01 – 169 Pseudomonas putida 23974 Pseudomonas putida IIB Salmonella enterica ‘typhimurium’ ATCC 14028 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 12228 Yersinia pseudotuberculosis ATCC 29833
Selective medium
Plasmid uptake
EC – blue BEA – no growth PIA – no fluorescence MSA – no growth None – unique morphology EC – purple EC – purple EC – purple EC – purple BEA – growth/brown None – unique morphology PIA – fluorescence PIA – fluorescence
–
PIA – fluorescence PIA – fluorescence EC – no pigment
– – –
– + + – – – – –
MSA – growth/yellow – MSA – growth/white – None – unique – morphology
Medium: EC = E. coli; BEA = bile esculin azide; PIA = Pseudomonas isolation agar; MSA = mannitol salt agar.
131
Fig. 1. Southern hybridization showing the presence of the qnrS gene on the donor and transconjugant strains located on a band migrating at approximately 150 kb.
cin would be useful for some strains but other strains had MICs just below 1 ng/μl. Putative transconjugants were restreaked to LB + 5 ng/ μl norfloxacin, and persistent colonies were screened by PCR for the presence of qnrS and qepA using the primers and methods detailed previously [Jung et al., 2009; Liu et al., 2008]. A positive transconjugant clone from each strain was further validated by Southern hybridization and through assessment of the antimicrobial response. A DIG-High Prime-labeled probe (Roche, Indianapolis, Ind., USA) of a qnrS PCR amplicon was generated and used to probe genomic preparations of transconjugants and donor that had been transferred from gels of pulsed field gel electrophoresis to nylon membranes, as detailed previously [Indest et al., 2010; Jung et al., 2011]. The conditions for the pulsed field gel electrophoresis were as follows: 0.5× TBE at 5 V/cm with a switch time of 30–90 s at a 120° angle for 24 h at 10 ° C for total DNA and 0.5× TBE at 5 V/cm with a switch time of 30–60 s at a 120° angle for 21 h at 10 ° C for 40 U I-CeuI/plug overnight digests of DNA (New England Biolabs, Ipswich, Mass., USA). The Saccharomyces cerevisiae and Lambda ladder markers (BioRad) were used as size standards. Southern hybridization revealed the presence of the qnrS gene on the donor and transconjugant strains located on a band migrating at approximately 150 kb, assuming linearization of the plasmid (fig. 1). MICs were performed for antimicrobials that had corresponding resistance genes on the strain YS11 plasmid [Park et al., 2009], like rmtB, which confers aminoglycoside resistance, blaTEM-1 and blaLAP-1, which confer β-lactamase resistance, and qnrS and qepA, which confer FQ resistance (table 2). E-Test strips (bioMerieux, Inc., Durham, N.C., USA) were used to establish MICs for am
132
J Mol Microbiol Biotechnol 2014;24:130–134 DOI: 10.1159/000362278
picillin, gentamicin, and chloramphenicol, while broth dilution MICs [Jung et al., 2009] were employed for the other antimicrobials. As anticipated, the transconjugants exhibited less sensitivity than the parent strains to each of the antimicrobials tested. However, the levels of resistance conferred far exceeded those expected for both qnrS and qepA genes, which may decrease sensitivity to ciprofloxacin by around 100-fold [Park et al., 2009], whereas our results show a 500- to 750-fold decrease. Previous work with E. aerogenes strain YS11 demonstrated that although aac(6′)-Ib is present in the donor strain YS11, it was not transferred to the E. coli J53 transconjugant [Park et al., 2009]. It was not made clear if the gene was located on another plasmid or chromosome, but in the present study the gene was not transferred (data not shown). Plasmid stability in transconjugants was assessed following the method of De Gelder et al. [2007]. Briefly, three colonies were separately inoculated from LB agar with 5 ng/μl norfloxacin into 5 ml LB broth and incubated at 37 ° C with shaking at 150 rpm. The cultures were transferred every 12 h (50 μl into 5 ml LB; 1:100 dilution) and appropriate dilutions were spread on LB plates. Resultant colonies were replica-plated (30 for each sample) on LB and LB with 5 ng/μl norfloxacin. The number of generations per day (24 h) for each strain had been previously determined. The percentage of colonies retaining the plasmid was observed over time and successive generations (fig. 2). E. coli S17 was determined to have a rate of 0.48% plasmid loss/generation (R2 = 0.95), while the plasmid in E. coli J53 was determined to be more stable at a rate of 0.17% plasmid loss/generation (R2 = 0.50). Overall, with no selective pressure, both strains exhibited high stability rates and there is no clear justification for the disparity between strains since they are both commonly
Jung
Table 2. MIC values (ng/μl) for the donor, recipient, and transconjugant strains
E. coli J53 Naladixic acid Ciprofloxacin Norfloxacin Enrofloxacin Lomefloxacin Levofloxacin Ampicillin Carbenicillin Gentamicin Kanamycin Streptomycin Chloramphenicol a
8 0.016 0.0625 0.125 1 0.0625 2 16 0.094 8 8 4
E. coli J53 Tran >256 (>32)a 12 (750) 64 (1,024) 2 (16) 8 (8) 1 (16) >256 (>128) >256 (>16) 256 (2,723) >256 (>32) 32 (4) 16 (4)
E. coli S17 64 0.004 0.0625 0.0156 1 0.0156 1.5 16 0.023 1 32 2
E. coli S17 Tran 128 (2) 2 (500) 64 (1,024) 0.25 (16) 8 (8) 1 (64) >256 (>170) >256 (>16) 64 (2,782) >256 (>256) 64 (2) 8 (4)
E. aerogenes YS11 >256 >32 64 4 32 16 >256 >256 >256 >256 256 24
E. coli 25922b 4 0.094 0.125 0.125 0.0625 0.0156 3 32 0.125 8 4 3
Fold changes from recipient to transconjugant are given in parentheses. b Antimicrobial-sensitive control strain.
used for cloning and mating and have similar genotypes with regard to uptake and maintenance of foreign DNA. This simple mating experiment has shown that there are aspects of HGT that are largely unknown. For instance, only two strains, both E. coli, appeared capable of taking up the IncF plasmid of strain YS11, while no transfer of the strain LR09 IncF plasmid was observed. The literature on the topic of HGT, in general, is equally vague with conflicting accounts of both stringent and highly promiscuous donor-recipient relationships. Indeed, De Gelder et al. [2005, 2007] showed that even in similar bac-
terial systems with a possible history of related mobile genetic elements (MGEs), the uptake and stability of MGEs was not predictable. Clearly, a more broad-based survey of mating pairs is needed for further understanding the global dissemination of MGEs. FQ resistance is ever increasing and there seems to be little oversight on the clinical usage of FQs as they are most often prescribed by primary care physicians [Dalhoff, 2012]. Multiple resistance mechanisms are most often found in the more resistant strains, and this is specifically true of the FQs [Hopkins et al., 2005; Robicsek et al., 2006]. An alarming trend toward co-selection of FQ resistance with other antimicrobials has been recently shown, whereby stool samples from patients administered non-FQs for 7 days showed a significant increase in FQ-resistant bacterial populations [Vien et al., 2012]. There is no easy answer for how to combat rampant resistance but it may be necessary to look at the other end usage, toward more judicious disposal of antimicrobials. In the absence of selective pressure many plasmids may not be sustainable for their host cell, so perhaps environmental decontamination of antimicrobial residues combined with more discriminate antimicrobial use should be the focus for reducing widespread antimicrobial resistance. Continual production of new antimicrobials without strict guidelines for usage or disposal repeatedly has led to circular path of excessive use due to success, followed by widespread resistance, further necessitating new antimicrobial development. Currently, pharmaceuticals are not treated as environmental contaminants [Cummings et al., 2011] by the EPA and are largely un-
Dissemination of Bacterial Fluoroquinolone Resistance
J Mol Microbiol Biotechnol 2014;24:130–134 DOI: 10.1159/000362278
Plasmid retention (%)
100 80 60 40 20 0 0
50
Generations
100
150
Fig. 2. Percent plasmid retention observed over time and successive generations.
133
regulated in drinking water (http://water.epa.gov/scitech/drinkingwater/dws/ccl/ccl3.cfm). It is estimated that 50 million pounds of antibiotics are produced each year, 25 million pounds of which are prescribed for human use, 300,000 pounds are sprayed on high-value crops, and millions of pounds of antibiotics are fed to animals every year (http://www.actionbioscience.org/ evolution/meade_callahan.html). In the USA, many of these compounds end up in the wastewater treatment plant environment as contaminants that are subsequently exposed to large numbers of diverse bacteria. The inevitable outcome of this cohabitation is the environmental propagation of antimicrobial resistance through natural evolutionary processes and the transfer of MGEs from resistant to susceptible bacteria via HGT. Reduction, not
obliteration, of the microbial load is the final step in most wastewater treatment plant processes, resulting in the release of resistant microbes back to the environment. Perhaps a more prudent use of antimicrobials combined with a complete life-cycle assessment ending with the destruction of the compounds would help reduce widespread and rampant spread of these genes and proliferation of multidrug-resistant bacteria.
Acknowledgements This research was funded from the US Army Corps of Engineers Environmental Quality Research Program.
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Hopkins KL, Davies RH, Threlfall EJ: Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int J Antimicrob Agents 2005;25:358–373. Indest KJ, Jung CM, Chen HP, Hancock D, Florizone C, Eltis LD, Crocker FH: Functional characterization of pGKT2, a 182-kb plasmid containing the xplAB genes involved in the degradation of RDX by Gordonia sp. KTR9. Appl Environ Microbiol 2010;76:6329–6337. Jacoby GA: Mechanisms of resistance to quinolones. Clin Infect Dis 2005;41:S120–S126. Jacoby GA, Walsh KE, Mills DM, Walker VJ, Oh H, Robicsek A, Hooper DC: qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother 2006; 50:1178–1182. Jung CM, Crocker FH, Eberly JO, Indest KJ: Horizontal gene transfer (HGT) as a mechanism of dissemination of RDX-degrading activity among Actinomycete bacteria. J Appl Microbiol 2011;110:1449–1459. Jung CM, Heinze TM, Strakosha R, Elkins CA, Sutherland JB: Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant. J Appl Microbiol 2009; 106:564–571. Liu JH, Deng YT, Zeng ZH, Gao JH, Chen L, Arakawa Y, Chen ZL: Coprevalence of plasmidmediated quinolone resistance determinants QepA, Qnr, and AAC(6′)-Ib-cr among 16S rRNA methylase RmtB-producing Escherichia coli isolates from pigs. Antimicrob Agents Chemother 2008;52:2992–2993. Martínez-Martínez L, Pascual A, Jacoby GA: Quinolone resistance from a transferable plasmid. Lancet 1998;351:797–799. Park Y-J, Yu JK, Kim SI, Lee K, Arakawa Y: Accumulation of plasmid-mediated fluoroquinolone resistance genes, qepA and qnrS1 in Enterobacter aerogenes co-producing RmtB
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and class A β-lactamase LAP-1. Ann Clin Lab Sci 2009;39:55–59. Reisner A, Wolinski H, Zechner EL: In situ monitoring of IncF plasmid transfer on semi-solid agar surfaces reveals a limited invasion of plasmids in recipient colonies. Plasmid 2012; 67:155–161. Robicsek A, Jacoby GA, Hooper DC: The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis 2006;6:629– 640. Rodriquez-Martinez JM, Cano ME, Velasco C, Martinez-Martinez L, Pascuel A: Plasmidmediated quinolone resistance: an update. J Infect Chemother 2011;17:149–182. Schmitz F-J, Jones ME, Hofmann B, Hansen B, Scheuring S, Lückefahr M, Fluit A, Verhoef J, Hadding U, Heinz H-P, Köhrer K: Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC. Antimicrob Agents Chemother 1998; 42:1249–1252. Vien LTM, Minh NNQ, Thuong TC, Khuong HD, Nga TVT, Thompson KT, Campbell JI, de Jong M, Farrar JJ, Schultsz C, van Doorn HR, Baker S: The co-selection of fluoroquinolone resistance genes in the gut flora of Vietnamese children. PLoS One 2012; 7: 1– 7. Xu X, Wu S, Ye X, Liu Y, Shi W, Zhang Y, Wang M: Prevalence and expression of the plasmidmediated quinolone resistance determinant qnrA1. Antimicrob Agents Chemother 2007; 51:4105–4110. Yamane K, Wachino Ji, Suzuki S, Kimura K, Shibata N, Kato H, Shibayama K, Konda T, Arakawa Y: New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob Agents and Chemother 2007; 51: 3354– 3360.
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Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Published online: May 22, 2014
Dissemination of Bacterial Fluoroquinolone Resistance in Two Multidrug-Resistant Enterobacteriaceae Carina M. Jung Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, Miss., USA
Key Words Horizontal gene transfer · Conjugation · Fluoroquinolone
Abstract Bacterial resistance to antimicrobials has become one of the greatest challenges for clinical microbiologists and healthcare practitioners worldwide. Acquisition of resistance genes has proven to be difficult to characterize and is largely uncontrollable in the environment. Here we sought to characterize conjugal horizontal gene transfer of plasmid-encoded fluoroquinolone resistance genes from two strains of Enterobacteriaceae, one clinical and one from a municipal wastewater treatment plant environment. Conjugation was dissimilar between the two strains. Escherichia coli strain LR09, containing a plasmid with the aac(6′)-Ib-cr fluoroquinolone resistance gene, did not conjugate with any of the 15 strains tested, while Enterobacter aerogenes strain YS11 conjugated with two strains of E. coli. The resultant transconjugants were also dissimilar in their stability and potential persistence. The observations presented herein exemplify the difficulties in understanding and controlling the spread of antimicrobial resistance. Thus, it may be prudent to address drug disposal and destruction, incorporating a life-cycle assessment plan ‘from cradle to grave’, treating antimicrobials as chemical or environmental contaminants. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1464–1801/14/0242–0130$39.50/0 E-Mail [email protected] www.karger.com/mmb
Bacterial resistance to fluoroquinolone (FQ) antimicrobials has become an increasingly serious issue over the past 10 years. Chromosomal point mutations in the quinolone resistance-determining region of DNA gyrase/ topoisomerase IV responsible for blocking the binding of FQs were first recognized in the 1980s [Dalhoff, 2012; Jacoby, 2005; Schmitz et al., 1998]. Efflux pump activity was then noted as a mechanism to decrease the effective concentration of the drug in the cell [Baucheron et al., 2004; Hopkins et al., 2005]. These chromosomal mutations, although heritable, are not generally considered to be disseminated to a larger population. However, later observations led to the discovery of plasmid-derived, transferable resistance mechanisms. The qnr system of pentapeptide repeat molecules serves to mimic the DNA gyrase/topoisomerase IV and reduces the effective intracellular concentration of the drug [Hata et al., 2005; Hegde et al., 2005; Jacoby et al., 2006; Martínez-Martínez et al., 1998]. The aac(6′)-Ib-cr gene is responsible for direct modification, through N-acetylation of the piperazine moiety of many FQs [Domagala, 1994; Jung et al., 2009], while QepA, a plasmid-associated efflux pump, clears FQs from the cell [Park et al., 2009; Yamane et al., 2007]. Views, opinions, and/or findings contained in this paper are those of the authors and should not be construed as an official Department of the Army position or decision unless so designated by other official documentation.
Carina M. Jung CEERD-EP-P US Army Engineer Research and Development Center 3909 Halls Ferry Road, Vicksburg, MS 39180 (USA) E-Mail Carina.M.Jung @ usace.army.mil
Table 1. Bacterial strains and selective media used in conjugation
Many characterized plasmids carrying FQ resistance genes have numerous transposable elements and similarities in various regions to other plasmids [Park et al., 2009; Rodriquez-Martinez et al., 2011]. It can be assumed that these plasmids are mobile and have gone through recombination events in their environment. We were interested in determining the prevalence of horizontal gene transfer (HGT) from bacterial strains harboring FQ-resistant plasmids to other bacteria of various lineages and conducted mating tests with two multidrug FQ-resistant Enterobacteriaceae. Escherichia coli strain LR09 [Jung et al., 2009], a wastewater treatment plant isolate from Little Rock, Arkansas, and Enterobacter aerogenes strain YS11 [Park et al., 2009], which is a clinical isolate from South Korea, were employed as plasmid (IncF) donors. Strain LR09 is resistant to FQs, β-lactams, aminoglycosides, and chloramphenicol. FQ resistance is conferred through mutations in the gyrA and parC genes of the quinolone resistance-determining region, active efflux mechanism(s), and a plasmid-encoded aac(6′)-Ib-cr gene [Jung et al.,
2009]. Strain YS11 contains a plasmid with qnrS1 and qepA FQ resistance genes as well as the genes for β-lactam and aminoglycoside resistance, blaTEM-1 and blaLAP-1, and rmtB, respectively. Mating recipients were first screened for sensitivity to norfloxacin at 1 ng/μl, then sodium azide (NaN3)-resistant mutants were generated as a means to counterselect against donors. A 25-μl drop of 100 μg/μl NaN3 was placed in the center of a spread plate of each culture and colonies growing closest to the NaN3 spot were passaged three times in Luria-Bertani (LB) broth + 100 ng/μl NaN3. A single NaN3-resistant mutant was isolated and used as a conjugation recipient strain for mating against FQ-resistant donors. E. coli strain J53 was already resistant to NaN3 and was excluded from mutant generation. Further, as a means to combat background NaN3-resistant donors, selective and differential media were used to isolate or differentiate transconjugants from donor strains (table 1). All media contained 100 ng/μl NaN3 and either 1 ng/μl or 5 ng/μl norfloxacin. Conjugal mating was established by growing overnight cultures of recipients in LB + 100 ng/μl NaN3 and donors in LB + 5 ng/μl norfloxacin. Cultures were pelleted and washed in sterile saline (0.85% NaCl). Donors were added to 5 ml LB at a final OD600 of 0.02, and recipients were added at a final OD600 of 0.2. Liquid mating in LB commenced for 24 h at 150 rpm on a platform shaker before the conjugation mixture was distributed to the appropriate selective plates (table 1). Various concentrations of donor:recipient (1:1, 1:2, 1:5, 1:10), addition of 1 ng/μl norfloxacin to the LB mating mixture, and filter mating were compared to the method listed above, but no measurable advantage was observed (data not shown). Differing total cell concentrations were not tested, although there is evidence that lower cell concentrations may increase mating efficiencies for IncF plasmids [Reisner et al., 2012]. Only two conjugal pairs, both recipients being E. coli strains, were observed with E. aerogenes strain YS11 (QepA+). The efficiency of conjugation for both YS11:J53 and YS11:S17 mating pairs were on the order of 10–8. No conjugation was observed with E. coli strain LR09. This may be due in part to the level of norfloxacin used in the screening, since aac(6′)Ib-cr potentially was the only FQ resistance gene being transferred. It is thought that aac(6′)-Ib-cr confers, at most, an around 10-fold increase in minimal inhibitory concentration (MIC) [Frasson et al., 2011; Xu et al., 2007]. If a recipient strain was extremely sensitive to norfloxacin, 1 ng/μl would have been too high of a screening level. Lowering the concentration of norfloxa-
Dissemination of Bacterial Fluoroquinolone Resistance
J Mol Microbiol Biotechnol 2014;24:130–134 DOI: 10.1159/000362278
trials Organism Donor Enterobacter aerogenes YS11
Recipients Bacillus cereus NCTR-466 Escherichia coli ATCC 47004 Escherichia coli strain J53 Escherichia coli strain S17 Escherichia coli ATCC 25922 Klebsiella pneumoniae ATCC 13883 Proteus mirabilis ATCC 7002 Pseudomonas fluorescens IC Pseudomonas aeruginosa ATCC PA01 – 169 Pseudomonas putida 23974 Pseudomonas putida IIB Salmonella enterica ‘typhimurium’ ATCC 14028 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 12228 Yersinia pseudotuberculosis ATCC 29833
Selective medium
Plasmid uptake
EC – blue BEA – no growth PIA – no fluorescence MSA – no growth None – unique morphology EC – purple EC – purple EC – purple EC – purple BEA – growth/brown None – unique morphology PIA – fluorescence PIA – fluorescence
–
PIA – fluorescence PIA – fluorescence EC – no pigment
– – –
– + + – – – – –
MSA – growth/yellow – MSA – growth/white – None – unique – morphology
Medium: EC = E. coli; BEA = bile esculin azide; PIA = Pseudomonas isolation agar; MSA = mannitol salt agar.
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Fig. 1. Southern hybridization showing the presence of the qnrS gene on the donor and transconjugant strains located on a band migrating at approximately 150 kb.
cin would be useful for some strains but other strains had MICs just below 1 ng/μl. Putative transconjugants were restreaked to LB + 5 ng/ μl norfloxacin, and persistent colonies were screened by PCR for the presence of qnrS and qepA using the primers and methods detailed previously [Jung et al., 2009; Liu et al., 2008]. A positive transconjugant clone from each strain was further validated by Southern hybridization and through assessment of the antimicrobial response. A DIG-High Prime-labeled probe (Roche, Indianapolis, Ind., USA) of a qnrS PCR amplicon was generated and used to probe genomic preparations of transconjugants and donor that had been transferred from gels of pulsed field gel electrophoresis to nylon membranes, as detailed previously [Indest et al., 2010; Jung et al., 2011]. The conditions for the pulsed field gel electrophoresis were as follows: 0.5× TBE at 5 V/cm with a switch time of 30–90 s at a 120° angle for 24 h at 10 ° C for total DNA and 0.5× TBE at 5 V/cm with a switch time of 30–60 s at a 120° angle for 21 h at 10 ° C for 40 U I-CeuI/plug overnight digests of DNA (New England Biolabs, Ipswich, Mass., USA). The Saccharomyces cerevisiae and Lambda ladder markers (BioRad) were used as size standards. Southern hybridization revealed the presence of the qnrS gene on the donor and transconjugant strains located on a band migrating at approximately 150 kb, assuming linearization of the plasmid (fig. 1). MICs were performed for antimicrobials that had corresponding resistance genes on the strain YS11 plasmid [Park et al., 2009], like rmtB, which confers aminoglycoside resistance, blaTEM-1 and blaLAP-1, which confer β-lactamase resistance, and qnrS and qepA, which confer FQ resistance (table 2). E-Test strips (bioMerieux, Inc., Durham, N.C., USA) were used to establish MICs for am
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picillin, gentamicin, and chloramphenicol, while broth dilution MICs [Jung et al., 2009] were employed for the other antimicrobials. As anticipated, the transconjugants exhibited less sensitivity than the parent strains to each of the antimicrobials tested. However, the levels of resistance conferred far exceeded those expected for both qnrS and qepA genes, which may decrease sensitivity to ciprofloxacin by around 100-fold [Park et al., 2009], whereas our results show a 500- to 750-fold decrease. Previous work with E. aerogenes strain YS11 demonstrated that although aac(6′)-Ib is present in the donor strain YS11, it was not transferred to the E. coli J53 transconjugant [Park et al., 2009]. It was not made clear if the gene was located on another plasmid or chromosome, but in the present study the gene was not transferred (data not shown). Plasmid stability in transconjugants was assessed following the method of De Gelder et al. [2007]. Briefly, three colonies were separately inoculated from LB agar with 5 ng/μl norfloxacin into 5 ml LB broth and incubated at 37 ° C with shaking at 150 rpm. The cultures were transferred every 12 h (50 μl into 5 ml LB; 1:100 dilution) and appropriate dilutions were spread on LB plates. Resultant colonies were replica-plated (30 for each sample) on LB and LB with 5 ng/μl norfloxacin. The number of generations per day (24 h) for each strain had been previously determined. The percentage of colonies retaining the plasmid was observed over time and successive generations (fig. 2). E. coli S17 was determined to have a rate of 0.48% plasmid loss/generation (R2 = 0.95), while the plasmid in E. coli J53 was determined to be more stable at a rate of 0.17% plasmid loss/generation (R2 = 0.50). Overall, with no selective pressure, both strains exhibited high stability rates and there is no clear justification for the disparity between strains since they are both commonly
Jung
Table 2. MIC values (ng/μl) for the donor, recipient, and transconjugant strains
E. coli J53 Naladixic acid Ciprofloxacin Norfloxacin Enrofloxacin Lomefloxacin Levofloxacin Ampicillin Carbenicillin Gentamicin Kanamycin Streptomycin Chloramphenicol a
8 0.016 0.0625 0.125 1 0.0625 2 16 0.094 8 8 4
E. coli J53 Tran >256 (>32)a 12 (750) 64 (1,024) 2 (16) 8 (8) 1 (16) >256 (>128) >256 (>16) 256 (2,723) >256 (>32) 32 (4) 16 (4)
E. coli S17 64 0.004 0.0625 0.0156 1 0.0156 1.5 16 0.023 1 32 2
E. coli S17 Tran 128 (2) 2 (500) 64 (1,024) 0.25 (16) 8 (8) 1 (64) >256 (>170) >256 (>16) 64 (2,782) >256 (>256) 64 (2) 8 (4)
E. aerogenes YS11 >256 >32 64 4 32 16 >256 >256 >256 >256 256 24
E. coli 25922b 4 0.094 0.125 0.125 0.0625 0.0156 3 32 0.125 8 4 3
Fold changes from recipient to transconjugant are given in parentheses. b Antimicrobial-sensitive control strain.
used for cloning and mating and have similar genotypes with regard to uptake and maintenance of foreign DNA. This simple mating experiment has shown that there are aspects of HGT that are largely unknown. For instance, only two strains, both E. coli, appeared capable of taking up the IncF plasmid of strain YS11, while no transfer of the strain LR09 IncF plasmid was observed. The literature on the topic of HGT, in general, is equally vague with conflicting accounts of both stringent and highly promiscuous donor-recipient relationships. Indeed, De Gelder et al. [2005, 2007] showed that even in similar bac-
terial systems with a possible history of related mobile genetic elements (MGEs), the uptake and stability of MGEs was not predictable. Clearly, a more broad-based survey of mating pairs is needed for further understanding the global dissemination of MGEs. FQ resistance is ever increasing and there seems to be little oversight on the clinical usage of FQs as they are most often prescribed by primary care physicians [Dalhoff, 2012]. Multiple resistance mechanisms are most often found in the more resistant strains, and this is specifically true of the FQs [Hopkins et al., 2005; Robicsek et al., 2006]. An alarming trend toward co-selection of FQ resistance with other antimicrobials has been recently shown, whereby stool samples from patients administered non-FQs for 7 days showed a significant increase in FQ-resistant bacterial populations [Vien et al., 2012]. There is no easy answer for how to combat rampant resistance but it may be necessary to look at the other end usage, toward more judicious disposal of antimicrobials. In the absence of selective pressure many plasmids may not be sustainable for their host cell, so perhaps environmental decontamination of antimicrobial residues combined with more discriminate antimicrobial use should be the focus for reducing widespread antimicrobial resistance. Continual production of new antimicrobials without strict guidelines for usage or disposal repeatedly has led to circular path of excessive use due to success, followed by widespread resistance, further necessitating new antimicrobial development. Currently, pharmaceuticals are not treated as environmental contaminants [Cummings et al., 2011] by the EPA and are largely un-
Dissemination of Bacterial Fluoroquinolone Resistance
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Plasmid retention (%)
100 80 60 40 20 0 0
50
Generations
100
150
Fig. 2. Percent plasmid retention observed over time and successive generations.
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regulated in drinking water (http://water.epa.gov/scitech/drinkingwater/dws/ccl/ccl3.cfm). It is estimated that 50 million pounds of antibiotics are produced each year, 25 million pounds of which are prescribed for human use, 300,000 pounds are sprayed on high-value crops, and millions of pounds of antibiotics are fed to animals every year (http://www.actionbioscience.org/ evolution/meade_callahan.html). In the USA, many of these compounds end up in the wastewater treatment plant environment as contaminants that are subsequently exposed to large numbers of diverse bacteria. The inevitable outcome of this cohabitation is the environmental propagation of antimicrobial resistance through natural evolutionary processes and the transfer of MGEs from resistant to susceptible bacteria via HGT. Reduction, not
obliteration, of the microbial load is the final step in most wastewater treatment plant processes, resulting in the release of resistant microbes back to the environment. Perhaps a more prudent use of antimicrobials combined with a complete life-cycle assessment ending with the destruction of the compounds would help reduce widespread and rampant spread of these genes and proliferation of multidrug-resistant bacteria.
Acknowledgements This research was funded from the US Army Corps of Engineers Environmental Quality Research Program.
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