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International Journal of Antimicrobial Agents 40 (2012) 455–457
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International Journal of Antimicrobial Agents
journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
Prevalence of quinolone resistance and mutations in the topoisomerase genes in
Salmonella enterica serotype Enteritidis isolates from Serbia
Gordana Kozoderović a, Maja Velhner b,∗, Zora Jelesić a, Nataša Golić c, Jelena Lozo c,d, Corinna Kehrenberg e
a
Institute of Public Health of Vojvodina, Futoška 121, 21000 Novi Sad, Serbia
Scientific Veterinary Institute ‘Novi Sad’, Rumenački put 20, 21000 Novi Sad, Serbia
c
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010 Belgrade, Serbia
d
Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
e
Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
b
a r t i c l e
i n f o
Article history:
Received 15 February 2012
Accepted 18 July 2012
Keywords:
Salmonella Enteritidis
gyrA
QRDR
Quinolones
Resistance
a b s t r a c t
The prevalence of quinolone resistance was studied in Salmonella enterica serotype Enteritidis isolates collected during 2005–2010 in Southern Bačka County, Serbia. A total of 878 clinical isolates were examined,
among which 19 (2.2%) nalidixic acid (NAL)-resistant S. Enteritidis were detected by selection on agar
plates containing 64 mg/L NAL. Antimicrobial susceptibility of the isolates was tested by the agar dilution method. According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, ciprofloxacin
(CIP) resistance was not present in the strains. Multiple drug resistance was rare, and resistance to NAL
was most often present as a single resistance property. All but one NAL-resistant S. Enteritidis showed
reduced susceptibility to CIP [i.e. minimum inhibitory concentration (MIC) ≥ 0.125 mg/L]. This isolate of
human origin had a CIP MIC of 0.064 mg/L and DNA sequencing revealed that it contained an Asp87Gly
gyrA mutation. Most of the remaining isolates had MICs for NAL and CIP of 256 mg/L and 0.256 mg/L,
respectively. Mutations in the Asp87 codon resulted in substitutions to Asn in most of the isolates, but
Asp87Gly and Ser83Phe exchanges were also detected. No mutations were present in the gyrB, parC or
parE genes. Although CIP resistance was absent, reduced susceptibility characterised by mutations in
gyrA was apparent among S. Enteritidis isolates from Serbia.
© 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
Salmonellae are one of the most important food-borne
pathogens. Owing to a high prevalence in livestock, they tend
to spread through the food chain, exposing humans to the risk
of salmonellosis. Clinical resistance to fluoroquinolones is rare,
but reduced susceptibility in Salmonella is an important concern since these antimicrobials are used to treat humans and
animals against severe Salmonella infections. The mechanisms of
quinolone resistance in Enterobacteriaceae are well documented.
The target enzymes for quinolones are DNA gyrase and topoisomerase IV, which are necessary for bacterial replication. Genes
encoding the DNA gyrase subunits are designated gyrA and gyrB,
whilst parC and parE encode topoisomerase IV. Reduced susceptibility to fluoroquinolones is mainly based on mutations in the
quinolone resistance-determining region (QRDR) of the gyrA gene,
which is the primary target of quinolone action. The QRDR of gyrA
∗ Corresponding author. Tel.: +38 121 489 5393; fax: +38 121 518 544.
E-mail addresses: [email protected], [email protected] (M. Velhner).
comprises a region from amino acid 67 to 106 [1], and the most
frequently detected mutations are at codons 83 and 87 [2]. Single
mutations normally cause resistance to nalidixic acid (NAL) and
decreased susceptibility to fluoroquinolones such as ciprofloxacin
(CIP). In addition, other mechanisms such as overexpression of multidrug efflux pumps or plasmid-mediated resistance genes like qnr,
aac(6 )-lb-cr and qepA may also contribute to decreased susceptibility [3,4].
In a previous investigation of a limited number of isolates, resistance to fluoroquinolones was not found, but 16.7% of Salmonella
enterica serotype Enteritidis from humans were resistant to NAL
[5]. The aim of the present study was to investigate the occurrence
of quinolone resistance in a larger collection of S. Enteritidis isolates
from human samples and to elucidate the mechanisms behind it.
Epidemiological aspects are briefly discussed.
2. Materials and methods
A total of 878 S. Enteritidis isolates of human origin were
selected from a strain collection of the Centre for Microbiology,
Institute of Public Health of Vojvodina (Novi Sad, Serbia). All isolates
0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
http://dx.doi.org/10.1016/j.ijantimicag.2012.07.012
Isolates were screened for resistance to NAL by striking 3 ␮L of
overnight broth cultures on Mueller–Hinton agar plates containing 64 mg/L NAL. Following 24 h of incubation, surviving colonies
were picked and tested for their susceptibility to a panel of selected
antibiotics.
2.2. Antimicrobial susceptibility testing
Minimal
inhibitory
concentrations
(MICs)
for
ampicillin, cefalothin, ceftriaxone, tetracycline, trimethoprim/sulfamethoxazole (SXT), NAL and CIP were determined
by the agar dilution method according to Clinical and Laboratory
Standards Institute (CLSI) guidelines [6]. Current CLSI breakpoints
were used for classifying the isolates as susceptible, intermediate
or resistant [7]. Escherichia coli ATCC 25922 was used for quality
control purposes.
2.3. PCR and sequencing
Overnight broth cultures were centrifuged and the pellets were
re-suspended in 200 ␮L of bidistilled water and subsequently
boiled for 5 min. PCR reactions were performed in a volume of 50 ␮L
using the Hot Start Kit from QIAGEN (Hilden, Germany). To detect
resistance-mediating mutations in the QRDRs of the target genes
gyrA, gyrB, parC and parE, the regions were amplified and sequenced
using previously described primer sets [3]. The cycling conditions
were as follows: initialisation at 94 ◦ C for 15 min; 30 cycles of denaturation at 94 ◦ C for 1 min, annealing at 55 ◦ C for gyrA, 58 ◦ C for gyrB
and 52 ◦ C for parC and parE for 1 min, and elongation at 72 ◦ C for
1 min; and final elongation for 10 min at 72 ◦ C. Purification of the
PCR products and sequencing on both strands were performed at
Macrogen (Amsterdam, The Netherlands).
3. Results and discussion
Salmonella Enteritidis is the most frequently detected serotype
among humans in Southern Bačka County, although a trend of
decrease in the total number of salmonellosis cases during recent
years was observed [8]. In this study, 19 S. Enteritidis isolates resistant to NAL were detected over a period of 6 years. None of these
isolates appeared to be clinically resistant to CIP. The isolates were
either resistant to NAL alone or in combination with resistance
to ampicillin and either cefalothin or SXT (Table 1). Multiple drug
resistance to three antimicrobial agents was detected in only two
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
parC
gyrB
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
AMP, ampicillin; CFT, cefalothin; CRO, ceftriaxone; TET, tetracycline; SXT, trimethoprim/sulfamethoxazole; NAL, nalidixic acid; CIP, ciprofloxacin.
a
MIC values indicative of resistance are shown in bold.
2.1. Screening for nalidixic-acid-resistant isolates of Salmonella
Enteritidis
Asp87Asn
Asp87Gly
Asp87Gly
Asp87Asn
Asp87Gly
Ser83Phe
Ser83Phe
Asp87Gly
Asp87Asn
Asp87Gly
Asp87Asn
Ser83Phe
Asp87Asn
Asp87Asn
Asp87Asn
Asp87Asn
Asp87Gly
Asp87Gly
Asp87Asn
were collected during the years 2005–2010 from Southern Bačka
County (Serbia). Isolates originated from hospitalised patients, outpatients and Salmonella carriers discovered during epidemiological
surveillance studies. Only one isolate per patient was included in
the study.
gyrA
trimetho-
0.256
0.256
0.256
0.256
0.256
0.256
0.512
0.256
0.256
0.064
0.256
0.256
0.256
0.256
0.256
0.256
0.125
0.125
0.256
SXT,
CIP
cefalothin;
NAL
CFT,
256
128
256
256
256
256
1024
128
256
128
256
128
512
512
256
256
256
256
256
ampicillin;
0.125/2.375
0.250/4.750
0.125/2.375
0.064/1.188
0.064/1.188
0.250/4.750
0.125/2.375
>4/76
0.500/9.500
0.250/4.750
0.064/1.188
0.064/1.188
0.125/2.375
0.125/2.375
0.125/2.375
0.125/2.375
0.125/2.375
0.125/2.375
0.125/2.375
3
–
–
SXT
2
–
–
2
4
4
1
1
2
2
2
4
1
1
1
1
1
2
1
1
1
1
–
–
–
TET
4
–
–
0.064
0.128
0.128
0.032
0.032
0.064
0.128
0.064
0.128
0.064
0.064
0.016
0.064
0.064
0.064
0.064
0.064
0.064
0.064
2
1
1
CRO
6
–
–
4
4
4
<1
2
4
4
8
4
16
2
2
4
4
4
2
2
4
4
2010
CFT
2009
2
4
2
0.5
2
2
4
>512
2
>512
2
2
2
2
1
2
2
2
2
2008
AMP
2007
Human stool, outpatient
Human stool, outpatient
Human stool, outpatient
Human stool, outpatient
Epidemiological surveillance
Hospitalised patient, clinic for infectious diseases
Human stool, outpatient
Human stool, outpatient
Human stool, outpatient
Hospitalised patient, paediatric clinic
Hospitalised patient, paediatric clinic
Human stool, outpatient
Human stool, outpatient
Hospitalised patient, paediatric clinic
Human stool, outpatient
Human stool, outpatient
Human stool, outpatient
Human stool, outpatient
Human stool, outpatient
NAL, nalidixic acid; AMP,
prim/sulfamethoxazole.
2006
182673/05
188487/05
202795/05
224725/05
260110/05
2989/06
120599/06
207096/06
238041/05
337167/06
91766/07
110574/07
138591/07
252107/07
54979/09
129066/09
122658/10
149323/10
248292/10
17
1
1
2005
QRDR mutations
Year of isolation
MIC (mg/L)a
NAL
AMP/CFT/NAL
AMP/SXT/NAL
Total no. of
isolates
Origin
Resistance
Isolate
Table 1
Resistance patterns in nalidixic acid-resistant Salmonella enterica serotype
Enteritidis.
parE
G. Kozoderović et al. / International Journal of Antimicrobial Agents 40 (2012) 455–457
Table 2
Origin of isolates, minimum inhibitory concentrations (MICs) and amino acid substitutions in the quinolone resistance-determining regions (QRDRs) of the topoisomerase genes for 19 nalidixic-acid-resistant Salmonella enterica
serotype Enteritidis isolates.
456
G. Kozoderović et al. / International Journal of Antimicrobial Agents 40 (2012) 455–457
isolates from 2006. The highest number of NAL-resistant S. Enteritidis was found in 2005, when six isolates were detected. In 2008
there was a conspicuous absence of NAL-resistant S. Enteritidis,
although the reason for this finding remains unclear. Nevertheless,
the overall frequency of resistance to nalidixic acid amounted to
2.2% and thus appears to be relatively low in Serbia compared with
other countries in East and West Europe. On the other hand, none
of the isolates tested appeared to be clinically resistant to CIP and
this finding is in good agreement with reports from most European
countries using CLSI breakpoints [9].
All of the NAL-resistant isolates contained mutations in the
QRDR region of the gyrA gene. Mutations in the other topoisomerase genes (gyrB, parC and parE) were not detected. MICs
of the 19 NAL-resistant S. Enteritidis ranged from 128 mg/L to
1024 mg/L for NAL and from 0.064 mg/L to 0.512 mg/L for CIP. The
most common combination of MIC values for NAL and CIP was
256 mg/L and 0.256 mg/L, respectively. Nearly all isolates showed
reduced susceptibility to CIP (MIC ≥ 0.125 mg/L) with the exception of one isolate that yielded a CIP MIC value of 0.064 mg/L [7].
However, this isolate possessed an Asp87Gly substitution in the
gyrA gene (Table 2). Similar findings have been reported previously. Hakanen et al. detected NAL-resistant Salmonella isolates
with single mutations in gyrA (Asp87Asn, Asp87Tyr or Asp87Gly)
that were also inhibited by 0.064 mg/L CIP [10]. In CIP-susceptible
Salmonella enterica serotype Typhimurium isolates from humans
and pigs in Taiwan (CIP MICs of 0.03–0.25 mg/L), single nucleotide
exchanges in gyrA were identified that led to amino acid exchange
at positions Ser83Phe or Ser83Tyr [11]. Furthermore, Cavaco and
Aarestrup [12] detected single mutations in the gyrA gene of S.
Enteritidis isolates of animal origin that also showed CIP MICs of
0.064 mg/L. In the isolates from Southern Bačka County, the most
frequent mutation was found in codon 87 and resulted in an amino
acid substitution Asp87Asn. This exchange was detected in 47.4%
(9/19) of the NAL-resistant isolates. Asp87Gly (7/19; 36.8%) and
Ser83Phe (3/19; 15.8%) exchanges were also detected. None of
the S. Enteritidis isolated during the 6-year time period in this
county contained a double gyrA mutation and this was in agreement with the MIC values for CIP (Table 2). Nevertheless, there was
no correlation between different gyrA mutations and MICs, meaning that each mutation type displayed various MICs for NAL and
CIP.
Resistance to quinolones is explained by a loss of interaction of
gyrase and quinolones caused by a substitution of the small polar
amino acid at 83 (Ser) with a large hydrophobic amino acid (Phe),
or substitution of the negatively charged Asp at position 87. In this
study, mutations in the QRDR of gyrA were found either in codon
83 or 87, as reported from other countries [13]. The fact that different mutation types appeared during the 6-year period in different
samples suggests that there was no clonal spread of NAL-resistant
S. Enteritidis.
457
The absence of clinical resistance and the low prevalence of
reduced susceptibility to CIP among S. Enteritidis of human origin are most likely attributed to the fact that layer flocks on Serbian
farms are not intensively treated with antibiotics. Since in Southern
Bačka County the most frequent sources of infection are homemade
meals with raw eggs, continuous education of the public sector is
very important to reduce the risk of food poisoning in the future.
Funding: This work was supported by grants from the Ministry
of Education and Science of the Republic of Serbia (project nos.
TR31071 and OI 173019).
Competing interests: None declared.
Ethical approval: Not required; isolates used in the research were
from an existing collection of isolates.
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