Pefloxacin as a Surrogate Marker for Fluoroquinolone Susceptibility for Salmonella typhi: Problems and Prospects
Balaji Veeraraghavan1, Shalini Anandan2, Dhiviya Prabaa Muthuirulandi Sethuvel3, Naveen Kumar Devanga Ragupathi4
1 Professor and Head, Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India.
2 Associate Professor, Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India.
3 Research Associate, Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India.
4 Senior Research Officer, Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India.
NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Balaji Veeraraghavan, Professor and Head, Department of Clinical Microbiology, Asha Building, 8th Floor, Christian Medical College, Vellore, Tamil Nadu, India.
E-mail: vbalaji@cmcvellore.ac.in
Sir,
The rise of MDR Salmonella typhi promoted the use of ciprofloxacin as the first line therapy since 2000 for enteric fever [1,2]. Due to selective pressure by extensive usage, there had been an emergence of resistance to ciprofloxacin. As per Clinical and Laboratory Standards Institute (CLSI), strains of Salmonella that test non-susceptible (intermediate), especially to ciprofloxacin, levofloxacin, ofloxacin, pefloxacin, or nalidixic acid may be associated with clinical failure or delayed response in fluoroquinolone-treated patients with salmonellosis [3]. Tests with nalidixic acid 30 μg and ciprofloxacin 5 μg disc will not reliably detect low-level resistance in Salmonella spp. Recently in 2015, CLSI recommended the use of 5 μg pefloxacin disc diffusion as a surrogate marker for identification of fluoroquinolone resistance in S.typhi [3]. This study was undertaken to evaluate the effectiveness of pefloxacin disc diffusion with ciprofloxacin disc diffusion and MIC breakpoints and highlights the problem and prospects of pefloxacin as surrogate marker.
Prospects of Pefloxacin
Earlier in 2012 the interpretative breakpoints for ciprofloxacin had been revised, where the susceptibility cut off using disc diffusion was raised from 21 to 31 mm and the MIC value was lowered from 1 to 0.06 μg/mL. In 2013, the disc diffusion interpretative criterion of levofloxacin and ofloxacin for S.typhi was removed. Meanwhile, the MIC interpretative criteria for levofloxacin and ofloxacin have been lowered to ≤0.12 μg/mL susceptible, 0.25-1 μg/mL intermediate and ≥2 μg/mL resistant. It is noteworthy that the interpretative criteria for ciprofloxacin, levofloxacin and ofloxacin have been changed only for typhoidal Salmonella in the Enterobacteriaceae family [4].
Recently (in 2015), CLSI and The European Committee on Antimicrobial Susceptibility Testing (EUCAST) have recommended the use of 5 μg pefloxacin disc diffusion test as reliable surrogate marker to identify the fluoroquinolone susceptibility to S.typhi [3,5]. Pefloxacin is understood to identify chromosomal (gyrA, gyrB, parC and parE); plasmid (qnrA, qnrB, qnrS and aac(6’)-lb-cr) mediated fluoroquinolone resistance better than nalidixic acid and ciprofloxacin (see [Table/Fig-1]). In addition, using pefloxacin can avoid the testing of ciprofloxacin and nalidixic acid by disc diffusion and MIC determination of levofloxacin, ofloxacin and ciprofloxacin [3].
Genotypic, phenotypic and quinolone correlation of resistance mechanism in Salmonella typhi.
| Genotype | Phenotype | Remarks |
|---|
| CLSI | CLSI | CLSI & EUCAST |
|---|
| Nalidixic acid(30 μg disc) | Ciprofloxacin(MIC - μg/ml) | | Pefloxacin(5 μg disc) |
|---|
| Chromosomal gyrA | Resistant | 0.12-1.0 | Nalidixic acid does not detect all mechanisms of fluoroquinolone resistance | Pefloxacin surrogate marker for all mechanism24 mm |
| Chromosomal gyrA and gyrB | Resistant | ≥4 |
| Plasmid qnrA, qnrB, qnrS and aac(6’)-lb-cr | Susceptible | 0.12-1.0 | - |
| No resistance gene | Susceptible | ≤0.06 | - | - |
Note: Limitation is that not all resistance mechanisms can be identified by a single test.
Pefloxacin, in Our Observation
Overall, 282 S.typhi isolates from community acquired blood stream infection were collected from January 2012 to December 2014 at Christian Medical College, Vellore, Tamil Nadu, India. All the isolates were tested for antimicrobial susceptibility by Kirby-Bauer disc diffusion method using ciprofloxacin (5 μg) and pefloxacin (5 μg), and E-test for ciprofloxacin. The results were interpreted according to the CLSI 2015 breakpoints and analysed.
Among the total 282, 4.3% (n = 12), 80.5% (n = 227) and 15.2% (n = 43) isolates were susceptible, intermediate and resistant respectively to ciprofloxacin by disc diffusion method. Similarly, 4.3% (n = 12), 80.5% (n = 227) and 15.2% (n = 43) isolates were susceptible, intermediate and resistant respectively to ciprofloxacin MIC breakpoints. Interestingly, 4.6% (n = 13) and 95.4% (n = 271) isolates were susceptible and resistant respectively to pefloxacin by disc diffusion.
According to CLSI interpretative criteria 80% (n = 225) of the isolates, intermediate to ciprofloxacin MIC fell under the category of resistant as per pefloxacin disc diffusion test. This observation perfectly matches with EUCAST ciprofloxacin MIC breakpoints as well. Further, a representative of 25 pefloxacin resistant isolates {ciprofloxacin MIC resistant (n = 14) and ciprofloxacin MIC intermediate (n = 11)} and two pefloxacin susceptible isolates were tested for gene mutations in gyrA, gyrB and parC genes. All the tested pefloxacin resistant isolates (n = 25) were observed to harbour gyrA and parC mutations (unpublished data).
Problems with Pefloxacin
The interpretative breakpoints defined both by CLSI and EUCAST for pefloxacin were narrow. The interpretative breakpoints for pefloxacin disc diffusion as per CLSI are ≤23 resistant and ≥24 susceptible, and <24 resistant and ≥24 susceptible by EUCAST. The chances for error are high with ± 1 mm difference in zone of inhibition by manual methods.
Since there is no gold standard to compare for pefloxacin disc diffusion testing, we have included a stringent quality control (QC) to avoid interpretation errors to a great extent. QC range for E. coli ATCC 25922 should be 25-31 mm, with a target of 28 mm and mean value of repeated tests for pefloxacin should be within 27-29 mm (target ± 1 mm) [6]. Also, one should be aware that the presence of inner colonies in pefloxacin disc diffusion testing suggests resistance [7]. In addition, pefloxacin cannot be used to detect the resistance mediated by aac(6’)-lb-cr, as this plasmid mediated mechanism is specific for fluoroquinolones possessing a piperazinyl secondary amine (ciprofloxacin and norfloxacin) [8].
The remarkable observation of 80% of the isolates, intermediate to ciprofloxacin MIC falls under the category of resistant as per peflo-xacin disc diffusion test (confirmed by molecular characterization). This clearly indicates that the patients with intermediate ciprofloxacin MIC if treated with high dose of ciprofloxacin will lead to treatment failure or delayed response. Conversely, if we consider pefloxacin as a surrogate marker for fluoroquinolone resistance, this will lead to appropriate interpretation and therapeutic success rate.
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