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J Antimicrob Chemother 2015; 70: 71 – 74 doi:10.1093/jac/dku320 Advance Access publication 21 August 2014 Polymorphism of genes encoding PmrAB in colistin-resistant strains of Escherichia coli and Salmonella enterica isolated from poultry and swine Alberto Quesada1*, M. Concepción Porrero2, Sonia Téllez2, Gonzalo Palomo1, Marı́a Garcı́a2 and Lucas Domı́nguez2 1 Departamento de Bioquı́mica, Biologı́a Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain; 2 Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense Madrid, Madrid, Spain *Corresponding author. Tel: +34927257000-1334; Fax: +34927257110; E-mail: [email protected] Received 4 June 2014; returned 9 July 2014; revised 14 July 2014; accepted 20 July 2014 Objectives: To detect the occurrence of low susceptibility to colistin (polymyxin E), a last-resort antimicrobial, among enterobacteria isolated from samples of animal origin (poultry and swine) and to find out the molecular basis of colistin resistance. Methods: Salmonella enterica and Escherichia coli were isolated from eggs and swine samples. Bacterial strains were screened for colistin resistance by using MIC determinations interpreted according to EUCAST recommendations. pmrAB genes were amplified by PCR from bacterial isolates and their sequences were characterized. Results: Nine colistin-resistant strains were detected in a collection of 739 enterobacteria (S. enterica and E. coli) isolated from animal samples taken in different environments. Sequences encoding the PmrAB two-component sensor – regulator from two colistin-resistant E. coli strains isolated from swine faeces presented three nonsynonymous polymorphisms, producing the variants 39SI and 81RS of PmrA and 161VG of PmrB, among which the involvement of mutations in PmrA-81 and PmrB-161 in resistance to the antimicrobial had been previously shown. No variation at the protein level was detected after analysis of PmrAB sequences from seven colistin-resistant S. enterica strains. Conclusions: E. coli strains carrying mutations in PmrAB that confer resistance to polymyxins, which might have evolved in vivo and have been rarely detected, are described for the first time in enterobacteria isolated from animals. Keywords: pmrAB genes, non-synonymous polymorphisms, colistin resistance, E. coli, S. enterica Introduction Polymyxins are cyclic pentapeptides, covalently bonded to a fatty acid chain, with strong antimicrobial activity against Gramnegative bacteria.1 The bactericidal action of polymyxins is mediated by their binding to the negatively charged lipopolysaccharide of the external membrane. Products of pmr (polymyxin resistance) genes promote the covalent modification of lipopolysaccharide, reducing the attachment of polymyxins to the external surface of Gram-negative bacteria.1 Since resistance to these antimicrobials is a rare event, polymyxin B and especially polymyxin E (colistin) are considered treatments of last resort against multidrug-resistant strains frequently found among nosocomial pathogens such as Klebsiella, Pseudomonas, Acinetobacter, Enterobacter and Escherichia coli.2 The genes pmrB and pmrA encode a two-component system with a sensor histidine kinase (PmrB) and its cognate regulator (PmrA), which, once phosphorylated, activates the expression of pmr genes.3 In vitro selection with colistin has been used to isolate mutants of PmrA and PmrB with up-regulated expression of pmr genes and colistin resistance in Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa and Acinetobacter baumannii.4 – 6 Moreover, polymorphisms of PmrA and/or PmrB of A. baumannii and Klebsiella pneumoniae have been detected in clinical isolates as well as after colistin treatment of human patients.7 – 10 Epidemiological surveillance of enterobacteria isolated worldwide from humans has provided evidence that colistin resistance remains low among E. coli (0.2%) and K. pneumoniae (1.5%), with no sign of an increase during 2006–09.11 Similarly, the prevalence of colistin resistance is low among isolates from animals, although there are exceptions, especially in clinical isolates from pigs in some countries.1 The fact that administration of colistin has become a regular practice in the treatment of colibacillosis # The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected] 71 Quesada et al. in poultry and swine production justified the recommendation for a permanent monitoring of colistin resistance among pathogenic bacteria. Materials and methods Isolates tested were recovered from surveillance programmes through the Spanish Surveillance Network of Antimicrobial Resistance in Bacteria of Veterinary Origin (VAV Network) and surveillance programmes for food of animal origin.12 Enterobacteria were isolated from eggs (sampled at retail, taken from different supermarkets) and swine samples (faeces from fattening pigs at slaughterhouses). MICs of antimicrobials were determined by using the 2-fold broth microdilution reference method according to ISO 20776– 1:2006. The threshold of antimicrobial resistance adopted in this work was based on the epidemiological cut-off (ECOFF) values recommended by EUCAST (http://www.eucast.org). The coding sequences of the pmrA and pmrB genes were amplified from crude DNA samples prepared by boiling cells in water. After centrifugation, the supernatant was used directly for PCR reactions with primers already described for Salmonella13 or with primers designed for E. coli pmrA [forward 5′ -AGTTTTCCTCATTCGCGACCA and reverse 5′ -TACCAGGC TGCGGATGATATTCT (product of 714 bp)] and E. coli pmrB [forward 5′ -GGATGGCCTGATGTGACGCTGTC and reverse 5′ -GCGCGGCTTTGGCTATA TGCTG (1312 bp)], by using annealing and extension at 688C. DNA fragments were purified from gels (Intron Biotechnology, Seongnam, South Korea) and sequenced by the STAB service of the University of Extremadura (Spain). Bioinformatics tools used in this work are publicly available at the web platforms of the NCBI (National Center for Biotechnological Information), EBI (European Bioinformatics Institute) and SMS (Sequence Manipulation Suite).14 Results Bacterial screening and antimicrobial resistance Enterobacteria isolated from eggs and swine samples were screened for colistin resistance using the ECOFF value defined by EUCAST (colistin MIC .2 mg/L; http://www.eucast.org). In these samples, taken during 2009 – 13, nine bacteria whose MICs of colistin were higher than ECOFF were detected (Table 1), seven corresponding to S. enterica (2.6% of total Salmonella isolates) and two to E. coli (0.4%). Susceptibility to other antimicrobials revealed that colistin resistance was not linked to a multidrug resistance phenotype, since only the E. coli isolate ZTA13/02182 presented the penta-resistant phenotype (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline), plus resistance to ciprofloxacin, gentamicin and kanamycin (Table 2). In contrast, the three Salmonella Enteritidis isolates shared only, in addition to colistin, low susceptibility to ciprofloxacin (Table 2). Polymorphism of pmrA and pmrB sequences among colistin-resistant isolates The coding sequences of the pmrA and pmrB genes were determined in bacteria screened for colistin resistance (Table 1), in the nine colistin-resistant (Table 2) plus nine randomly selected colistin-susceptible strains (colistin MIC ,2 mg/L), including one S. enterica isolated in 2013 (ZTA13/1700) and eight E. coli from 2011 (ZTA11/0606, ZTA11/1357, ZTA11/1860 and ZTA/2612) and 2013 (ZTA13/0455, ZTA13/1067, ZTA13/1622 and ZTA13/ 2284). The eight S. enterica isolates, seven colistin-resistant strains (Table 2) and one colistin-susceptible strain shared a total of 12 polymorphic positions that were all synonymous at the protein level (not shown). Moreover, PmrA and PmrB sequences deduced from S. enterica isolates in this work did not present any of the protein polymorphisms detected after in vitro selection of colistin-resistant strains in S. enterica.13 By contrast, seven out of the 22 polymorphisms detected in pmrA and pmrB coding sequences from the 10 E. coli isolates, two colistinresistant (Table 2) and seven colistin-susceptible strains, were non-synonymous (Figure S1, available as Supplementary data at JAC Online). Among these, polymorphisms that produce protein variants 144GS (PmrA), 283DG (PmrB) and 357YN (PmrB) were found only in colistin-susceptible strains (Figure S1) and did not match any mutation previously detected after in vitro selection with colistin.13 Moreover, none of the three non-synonymous polymorphisms found between the two colistin-resistant E. coli strains (Table 2) had previously been described in E. coli, as evidenced by BLASTn comparison against the sequence database no./nt (nucleotide collection, NCBI). Remarkably, two of the mutations uniquely found in colistin-resistant E. coli strains, 81RS of PmrA and 161VG of PmrB, have also been found after in vitro selection of S. enterica colistin-resistant strains.13 Discussion The fact that mutations of the PmrAB two-component sensor – regulator might confer polymyxin resistance on Gram-negative bacteria is a matter of the highest clinical relevance.1 However, despite exhaustive surveillance, enterobacteria presenting such polymorphisms had not been detected in nature before the present work, although a recent exception is represented by K. pneumoniae strains recovered from a human patient treated with colistin.11 Interestingly, among the 27 residues affected by nonsynonymous polymorphisms that were found after in vitro selection of colistin-resistant strains in Salmonella,13 positions R81 of PmrA and V161 of PmrB were also found mutated in non-clinical isolates of E. coli from swine faeces analysed in this work (Table 2). None of these two mutations has been detected before in Table 1. Bacterial screening for resistance to colistin Bacteria S. enterica S. enterica S. enterica E. coli 72 Source Sampling period Samples Isolates Number of colistin-resistant isolates eggshell swine faeces swine ganglia swine faeces 2013 2009 2010– 11 2011, 2013 500 284 474 487 9 99 160 471 3 1 3 2 JAC Polymorphism of pmrAB genes in colistin-resistant enterobacteria from animals Table 2. Antimicrobial resistance and PmrAB polymorphisms of colistin-resistant isolates Isolatea Year Identification Antimicrobial resistance Colistin MIC (mg/L) PmrABb ZTA13/01772 ZTA13/01834 ZTA13/01839 ZTA09/00762 ZTA10/00723 ZTA10/00996 ZTA11/01078 ZTA11/01748 ZTA13/02182 2013 2013 2013 2009 2010 2010 2011 2011 2013 Salmonella Enteritidis Salmonella Enteritidis Salmonella Enteritidis Salmonella Typhimurium Salmonella Typhimurium Salmonella Rissen Salmonella Typhimurium E. coli E. coli CIP CIP CIP PR (without CHL) PR PR PR+CIP+KAN PR (without CHL) PR+CIP+GEN+KAN 4 4 4 8 8 8 8 4 4 — — — — — — — 161VG (PmrB) 39SI+81RS (PmrA) CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; PR, penta-resistance; ECOFF and MIC distributions are available at www. eucast.org. a Isolation sources for strains were: eggshells (ZTA13/01772, ZTA13/01834 and ZTA13/01839), swine ganglia (ZTA10/00723, ZTA10/00996 and ZTA11/ 01078) and swine faeces (ZTA09/00762, ZTA11/01748 and ZTA13/02182). b Polymorphisms found for coding sequences for PmrA or PmrB. sequences from E. coli, even though .1000 pmrA and pmrB sequences from these bacteria are available for comparison. Considering that colistin resistance in E. coli occurs at extremely low frequency (www.eucast.org), most if not all E. coli pmrAB sequences deposited in databases would represent colistinsusceptible strains (with the exception of strains ZTA11/01748 and ZTA13/02182 in the present work), and thus the link found between the mutations and colistin resistance should be considered a highly significant association, although it was not observed for colistin resistance of S. enterica isolated in this work. Accordingly, colistin resistance in Salmonella spp. has recently been shown independently of PmrAB polymorphism in a large screening of bacteria from different environments, including samples from animals and their products, where it was shown that some genetic backgrounds, such as serovar Enteritidis, might express levels of colistin resistance even higher than the recommended ECOFF of 2 mg/L.15 Although the 81RS polymorphism of PmrA found in ZTA13/ 02182 does not match exactly the allelic variants detected previously in Salmonella (81RH and 81RC),13 including the first ever found in a polymyxin-resistant bacteria,4 it corresponds to a strictly conserved position of the protein.13 In addition, the 161VG mutation of PmrB found in ZTA11/01748 is the same polymorphism selected in vitro by colistin in Salmonella.12 Both mutations might affect phosphate transfer between the two components of the system since V161 is located in the kinase domain of PmrB, whereas R81 is in the phosphate acceptor domain of PmrA.13 The other unique polymorphism found among PmrA sequences in this work, 39SI in the ZTA13/ 02182 E. coli isolate, affects a residue that is not strictly conserved during protein evolution and has not been detected by in vitro selection in Salmonella.13 This contribution represents, to our knowledge, the first description of PmrAB polymorphisms in colistin-resistant strains of E. coli or any other enterobacteria isolated from the environment. Although the finding of only two colistin-resistant strains from animal samples (swine faeces), taken in different years (2011 and 2013), could be considered a rare event, the extensive use of colistin in animal production justifies the importance of surveying their appearance in nature, which could challenge the efficacy of polymyxins in controlling multidrug-resistant organisms. Nucleotide sequence accession numbers Sequences determined in this work have been deposited in the EMBL sequence database with accession codes from HG964672 to HG964681 and LK934653 to LK934660. Acknowledgements We wish to thank our technicians Estefania Rivero and Carolina Castilla for their excellent technical assistance. Funding This work was supported by the Ministry of Innovation, Science and Technology of Spain (AGL2012-39028-C03-01 and AGL2012-39028C03-03), the Department of Employ, Enterprise and Innovation of the regional government of Extremadura, Spain (Group CTS001), the University of Extremadura (Group MIVET), the Spanish Ministry of Agriculture, the Regional Government of Madrid and the Autonomous Community of Madrid (S2009/AGR-1489). Transparency declarations None to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/). References 1 Kempf I, Fleury MA, Drider D et al. What do we know about resistance to colistin in Enterobacteriaceae in avian and pig production in Europe? Int J Antimicrob Agents 2013; 42: 379–83. 73 Quesada et al. 2 Landman D, Georgescu C, Martin DA et al. Polymyxins revisited. Clin Microbiol Rev 2008; 21: 449– 65. 3 Gunn JS, Lim KB, Krueger J et al. PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol Microbiol 1998; 27: 1171– 82. 4 Roland KL, Martin LE, Esther CR et al. Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence. J Bacteriol 1993; 175: 4154 –64. 5 Moskowitz SM, Ernst RK, Miller SI. PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A. J Bacteriol 2004; 186: 575–9. 6 Adams MD, Nickel GC, Bajaksouzian S et al. Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB twocomponent system. Antimicrob Agents Chemother 2009; 53: 3628–34. 7 Arroyo LA, Herrera CM, Fernandez L et al. The pmrCAB operon mediates polymyxin resistance in Acinetobacter baumannii ATCC 17978 and clinical isolates through phosphoethanolamine modification of lipid A. Antimicrob Agents Chemother 2011; 55: 3743 –51. 8 Rolain JM, Diene SM, Kempf M et al. Real-time sequencing to decipher the molecular mechanism of resistance of a clinical pan-drug-resistant Acinetobacter baumannii isolate from Marseille, France. Antimicrob Agents Chemother 2013; 57: 592– 6. 74 9 Snitkin ES, Zelazny AM, Gupta J et al. Genomic insights into the fate of colistin resistance and Acinetobacter baumannii during patient treatment. Genome Res 2013; 23: 1155– 62. 10 Cannatelli A, Pilato VD, Giani T et al. In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC carbapenemase-producing Klebsiella pneumoniae associated with low-dosage colistin treatment. Antimicrob Agents Chemother 2014; 58: 4399– 403. 11 Gales AC, Jones RN, Sader HS. Contemporary activity of colistin and polymyxin B against a worldwide collection of Gram-negative pathogens: results from the SENTRY Antimicrobial Surveillance Program (2006 – 09). 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