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