JCM Accepted Manuscript Posted Online 19 August 2015 J. Clin. Microbiol. doi:10.1128/JCM.01287-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Development of a Pefloxacin Disk Diffusion Method for Detection of
2
Fluoroquinolone-resistant Salmonella Enterica
3 4
Robert Skova#, Erika Matuschekb, Maria Sjölund-Karlssonc, Jenny Åhmanb, Andreas Petersena, Marc Steggera,
5
Mia Torpdahla and Gunnar Kahlmeterb
6 7
a
Statens Serum Institut, Copenhagen (SSI), Denmark; bEUCAST Development Laboratory (EDL), Växjö, Sweden;
8
c
9
Atlanta, GA 30329, USA.
Division of Foodborne, Waterborne, and Environmental Diseases, Centers for Disease Control and Prevention,
10 11
Running Head: Pefloxacin Detection of FQ-resistant Salmonella
12 13 14 15
Corresponding author:
16
Robert Skov, Department of Microbiology and Infection Control, Statens Serum Institut, Artillerivej 5,
17
Copenhagen, 2300 Denmark
18
Phone +45 3268 8348, email
[email protected]
19
Abstract
20
Fluoroquinolones are among the drugs of choice for treatment of salmonella infections. However,
21
fluroquinolone resistance is increasing in salmonella due to chromosomal mutations in the quinolone
22
resistance-determining region (QRDR) of the topoisomerase genes gyrA, gyrB, parC and parE and/or plasmid-
23
mediated quinolone resistance (PMQR) mechanisms including qnr variants, aac(6’)-Ib-cr, qepA and oqxAB.
24
Some of these cause only subtle increases of the MIC i.e, MICs ranging from 0.12 – 0.25 mg/L for ciprofloxacin
25
(just above the wild type MIC of ≤0.06 mg/L). These isolates are difficult to detect with standard ciprofloxacin
26
disk diffusion, and plasmid mediated resistance such as qnr are often not detected by the nalidixic acid screen
27
test. We evaluated 16 quinolone/fluoroquinolone disks for their ability to detect low-level resistant Salmonella
28
non-typhi isolates. A total of 153 Salmonella isolates characterized for the presence (N=104) or absence (N=49)
29
of gyrA/parC topoisomerase mutations, qnrA, qnrB, qnrD, qnrS, aac(6’)-lb-cr or qepA genes were investigated.
30
All isolates were MIC tested by broth micro-dilution against ciprofloxacin, levofloxacin and ofloxacin and by
31
disk diffusion using EUCAST/CLSI methodology. MIC determination correctly categorized all isolates as either
32
wildtype (MICs ≤0.06 mg/L and absence of resistance genes) or non-wildtype (MIC >0.06 mg/L and presence of
33
a resistance gene). Disk diffusion using these antibiotics and nalidixic acid failed to detect some low-level resistant
34
isolates whereas the pefloxacin 5 µg disk correctly identified all resistant isolates. However, pefloxacin will not
35
detect isolates having aac(6’)-Ib-cr as the only resistance determinant. The pefloxacin disk assay was approved
36
and implemented by EUCAST(2014) and CLSI(2015).
37
Introduction
38
Human infections caused by Salmonella enterica subspecies enterica represent a major burden worldwide (1).
39
Typhoidal Salmonella (Salmonella Typhi and Paratyphi A) cause enteric fever, a severe systemic and febrile
40
illness, whereas non-typhoidal serotypes primarily cause self-limiting diarrhoea with occasional bacteraemia.
41
Timely treatment with antimicrobial agents is critical for optimal treatment of both enteric fever and invasive
42
non-typhoidal Salmonella infections. Fluoroquinolones (e.g. ciprofloxacin) are highly efficient against fully
43
susceptible (i.e. isolates without any resistance mechanisms) whereas their efficacy is in doubt as soon as any
44
resistance can be detected and there is concern about the rapidly increasing fluoroquinolone resistance in
45
Salmonella (2-5).
46
Fluoroquinolone resistance in Salmonella is mainly caused by chromosomal mutations in the
47
quinolone resistance-determining region (QRDR) of the topoisomerase genes gyrA, gyrB, parC and parE (6,7).
48
These mutations usually confer stepwise resistance; a single mutation is associated with a ciprofloxacin
49
minimum inhibitory concentration (MIC) of 0.12-0.5 mg/L whereas two or more mutations result in higher MIC
50
values. Topoisomerase mutations are associated with resistance to the quinolone nalidixic acid (MIC >16 mg/L)
51
(6).
52
In addition to the QRDR topoisomerase mutations, a number of plasmid-mediated quinolone
53
resistance (PMQR) mechanisms have been described including qnr variants, aac(6’)-Ib-cr, qepA and oqxAB, with
54
qnr genes being the predominant PMQR mechanism among Salmonella (7,8).
55
The PMQR mechanisms result in reduced susceptibility to ciprofloxacin (MIC 0.125-1.0 mg/L) but only a modest
56
or no increase in nalidixic acid (MIC 8-32mg/L) (8). Although the PMQR mechanisms only confer a moderate
57
increase in fluoroquinolone MICs, they are clinically relevant; patients infected with both Salmonella Typhi and
58
non-typhoidal Salmonella isolates with ciprofloxacin MICs of 0.125-1.0 mg/L have more treatment failures and
59
longer times to fever clearance than patients with isolates fully susceptible to ciprofloxacin (MIC ≤ 0.06 mg/L)
60
(2, 9-11).
61
Since Salmonella isolates with low-level fluoroquinolone resistance may be associated with
62
failed response to fluoroquinolone treatment, it is important that these isolates are detected during routine
63
antimicrobial susceptibility testing. This has been recognized by the Clinical and Laboratory Standards Institute
64
(CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the two international
65
bodies providing antimicrobial susceptibility testing guidelines. Initially, both EUCAST and CLSI included
66
warnings for using fluoroquinolones for treating infections caused by Salmonella spp. with ciprofloxacin MICs >
67
0.06 mg/L and later both organizations introduced species-specific MIC breakpoints for Salmonella and
68
fluoroquinolones to aid in the detection of isolates with acquired resistance (12-14).
69
For laboratories using disk diffusion, detection of low-level resistant isolates is challenging. With
70
ciprofloxacin, an overlap in inhibition zone diameters between wild type isolates and isolates with low-level
71
resistance has been reported for the 5 µg disk (15-17). For many years, both CLSI and EUCAST therefore
72
recommended the use of a nalidixic acid disk as a screening test. However, this does not adequately detect
73
isolates with PMQR, which are often susceptible to nalidixic acid (MIC ≤16 mg/L) (8).
74 75
The aim of this study was to investigate whether or not one of 16 evaluated fluoroquinolone disks could identify all known fluoroquinolone resistance mechanisms in Salmonella isolates.
76
Materials and Methods
77
Bacterial isolates
78
A total of 153 non-typhoidal Salmonella isolates were included, 98 from Statens Serum Institut (SSI),
79
Copenhagen, Denmark and 55 through the National Antimicrobial Resistance Monitoring System (NARMS) at
80
the Centers for Disease Control and Prevention (CDC), Atlanta, USA (Supplementary table 1). To ensure a high
81
proportion of isolates exhibiting difficult-to-detect low-level fluoroquinolone resistance, the collection
82
consisted of 104 isolates with ciprofloxacin MICs in the range of 0.125-0.5 mg/L and 49 isolates fully susceptible
83
to nalidixic acid and ciprofloxacin (MIC ≤0.064 mg/L). Serotype distribution was very similar in the two
84
materials.
85 86
DNA isolation, PCR amplification and Sequencing
87
All 153 isolates were investigated for the presence of gyrA/parC topoisomerase mutations, qnrA, qnrB, qnrD,
88
qnrS, aac(6’)-lb-cr, and qepA genes. For each isolate, crude genomic DNA was prepared by lysing the bacteria at
89
95°C for 10 minutes and collecting the supernatant after a brief centrifugation. The presence of these genes
90
and mutations were investigated by PCR and Sanger sequencing using the following primer pairs (5’ to 3´); parC
91
(CTATGCGATGTCAGAGCTGG, TAACAGCAGCTCGGCGTATT) (18), gyrA
92
(ATGAGCGACCTTGCGAGAGAAATTACACCG, TTCCATCAGCCCTTCAATGCTGATGTCTTC) (19), qnrA
93
(GGATGCCAGTTTCGAGGA, TGCCAGGCACAGATCTTG) (20), qnrB (GGMATHGAAATTCGCCACTG,
94
TTTGCYGYYCGCCAGTCGAA) (21), qnrD (CGAGATCAATTTACGGGGAATA, AACAAGCTGAAGCGCCTG) (22), qnrS
95
(TCGACGTGCTAACTTGCG, GATCTAAACCGTCGAGTTCGG) (20), aac(6’)-Ib-cr (TTGCGATGCTCTATGAGTGGCTA,
96
CTCGAATGCCTGGCGTGTTT) (23), and qepA (TGGTCTACGCCATGGACCTCA, TGAATTCGGACACCGTCTCCG) (24).
97
Each PCR was performed in a 100 µl volume using AmpliTaq (Perkin Elmer, Waltham, MA, USA) on a GeneAmp
98
PCR System 2400 (Perkin Elmer). Amplicons were purified using QIAquick PCR Purification Kit (QIAGEN,
99 100
Valencia, CA, USA) according to the manufacturer’s instructions, and subsequently sequenced on an ABI PRISM 373 DNA Sequencer (PE Applied Biosystems, Foster City, CA, USA).
101 102
Antimicrobial Susceptibility Testing
103
MICs for ciprofloxacin, levofloxacin, nalidixic acid and ofloxacin were determined using broth microdilution
104
(BMD) according to the ISO standard 20776-1. Testing was performed using customized frozen (TREK
105
Diagnostics/Thermo Fisher Scientific, Oakwood, OH) or lyophilized (TREK Diagnostics/Thermo Fisher Scientific,
106
Basingstoke, United Kingdom) panels.
107
Disk diffusion (DD) was performed according to EUCAST and CLSI methodology i.e. Mueller
108
Hinton (MH) agar medium, inoculum of 0.5 McFarland taken from fresh overnight cultures and incubation for
109
16-20 hours at 35°C in ambient air (25,26).
110
E. coli ATCC 25922 was used as quality control strain in all assays.
111 112
Identification of candidate disks suitable for screening for fluoroquinolone resistance
113
Sixteen Fluoroquinolone and quinolone disks (Table 1) were evaluated for their ability to detect low-level
114
fluoroquinolone resistance in Salmonella enterica. This work was performed at the EUCAST Development
115
Laboratory Växjö, Sweden (EDL) and at the Reference Laboratory for Antibiotic Resistance at Statens Serum
116
Institut (SSI), Copenhagen, Denmark. For this evaluation, 87 of the 153 isolates were included and tested on
117
two brands of Mueller-Hinton (MH) media (BBL, Becton Dickinson, Baltimore, US and Oxoid, Thermo Fisher
118
Scientific, Basingstoke, United Kingdom). All tests were read by two persons, giving four readings per isolate,
119
i.e. a total of 87 isolates x 2 media x 2 persons = 348 readings per disk. Four disks were included in a second
120
round of testing (ciprofloxacin 1 µg, enoxacin 10 µg, norfloxacin 2 µg and pefloxacin 5 µg) along with
121
ciprofloxacin (5 µg), nalidixic acid (30 µg), levofloxacin (5 µg), and ofloxacin (5 µg) disks. This second evaluation
122
was performed at three laboratories (EDL, SSI and NARMS-CDC) and included 126 isolates (Table 2). All three
123
sites used the same lots of disks and performed testing on two different types of MH media using commercial
124
BBL Mueller-Hinton agar plates (Becton Dickinson) as common media. In addition, NARMS-CDC used
125
commercial plates from Remel (Thermo Fisher Scientific, Waltham, MA, USA), EDL used in-house prepared
126
plates based on Mueller-Hinton powder from Oxoid and SSI used in-house prepared plates based on BBL MH
127
powder from Becton Dickinson. Altogether, this resulted in 126 isolates x 3 laboratories x 2 media = 756
128
readings for each disk. The purpose of using different brands of media and different readers were to
129
investigate the robustness of the method by simulating the everyday situation where media from different
130
manufacturers would be used in many laboratories and where results would depend on the acuity of many
131
readers. The results were therefore interpreted collectively rather than by individual media and reader.
132 133
Validation of the pefloxacin 5 µg disk
134
Once we had chosen to develop the method on the pefloxacin disk, two steps were taken to validate the disk
135
diffusion assay. As a first step, differences in pefloxacin disk potency among disks from different manufacturers
136
was assessed by testing 24 selected isolates against pefloxacin 5 µg disks from Becton Dickinson, Bio-Rad
137
(Marnes-la-Coquette, France), MAST Diagnostic (Bootle,Merseyside, UK) and Oxoid. The disk potency was
138
investigated independently at two sites using a bioassay (27).
139
Secondly, pefloxacin 5 µg disks (Oxoid) were evaluated on consecutive clinical isolates as part of
140
the routine disk diffusion testing at the department of Clinical Microbiology, Kronoberg and Blekinge counties,
141
Sweden, alternating between Mueller-Hinton agar from BD, Bio-Rad and Oxoid and ten different readers.
142
Results
143
A fluoroquinolone resistance mechanism was identified in all 104 ciprofloxacin non-wild type isolates
144
(ciprofloxacin MIC >0.06 mg/L); 53 harboured a topoisomerase mutation in gyrA, 50 harboured a qnr gene and
145
a single isolate harboured the aac(6)’lb-cr gene. The corresponding MIC values for levofloxacin were >0.125
146
mg/L and for ofloxacin >0.125 mg/L while for nalidixic acid, isolates with a topoisomerase mutation all
147
displayed nalidixic MIC of >16 mg/L whereas isolates with a plasmid-mediated mechanism exhibited MIC values
148
of 4->64 mg/L (Supplementary table 1).
149
In the 49 isolates with ciprofloxacin MICs ≤0.06 mg/L, there were no fluoroquinolone resistance
150
mechanisms (gyrA/parC, qnr, qep or aac(6)’lb-cr genes). The corresponding MIC values were ≤0.125 mg/L for
151
levofloxacin and ofloxacin and ≤16 mg/L for nalidixic acid. Thus, with standardized broth micro dilution MIC
152
determination, ciprofloxacin, levofloxacin and ofloxacin, accurately and equally well, categorized the 49
153
isolates as belonging to the wild type.
154 155
For E. coli ATCC 25922, all MIC values for ciprofloxacin (N=23), levofloxacin (N=13), ofloxacin (N=18) and nalidixic acid (N= 18) were within the established QC ranges (13,28).
156 157
Identification of candidate disks suitable for screening for fluoroquinolone resistance
158
The results for each of the 16 disks are shown in Table 1. Since there was a perfect correlation between MICs
159
and resistance mechanisms for all three fluoroquinolones, disk diffusion results were correlated to
160
ciprofloxacin MIC values and resistance mechanisms throughout. Our results confirmed that the nalidixic acid
161
30 µg disk is reliable only for the detection of isolates with topoisomerase mutations. A considerable overlap,
162
and thus poor distinction, between wild type and non-wild type isolates was also observed for the ciprofloxacin
163
5 µg, levofloxacin 5 µg and ofloxacin 5 µg disks. In contrast, the ciprofloxacin 1 µg, enoxacin 10 µg, norfloxacin
164
2 µg and pefloxacin 5 µg disks were able to distinguish between wild type and non-wild type isolates and were
165
for this reason considered for the next stage of the study where three laboratories (EDL, SSI and CDC) were
166
involved in the evaluation (Table 2).
167
Again, excellent results were obtained with the pefloxacin 5 µg disk - only two of 756 readings
168
(0.3%) resulted in overlapping zone diameters between isolates with and without a resistance mechanism.
169
When each of the MH agars was analysed separately, clear separation between wild type and non-wild type
170
isolates was achieved on all media (Supplementary Figure 1a-f). This was not the case with the ciprofloxacin 5
171
µg disk (Supplementary Figure 1g-l).
172
As shown in Tables 1 and 2, ciprofloxacin 1 µg, enoxacin 10 µg and norfloxacin 2 µg disks also
173
performed well. Neither of the disks performing well were part of the CLSI and EUCAST standard panel of
174
disks. Pefloxacin was chosen for further development because of its slightly better performance on the
175
individual brands of Mueller Hinton media (data not shown) and because it was available from at least four
176
manufacturers. Based on the initial disk diffusion results, a tentative screening breakpoint of wild type ≥24 mm
177
and non-wild type of
