Download Polymorphism of genes encoding PmrAB in colistin

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.
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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/).
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