Development of a Pefloxacin Disk Diffusion Method for Detection of ...

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Aug 19, 2015 - mediated quinolone resistance (PMQR) mechanisms including qnr variants, ... Disk diffusion using these antibiotics and nalidixic acid failed to ...
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