Rapid and Simple Determination of Ciprofloxacin Resistance in. Clinical Strains of Escherichia coli

Transcription

1 JCM Accepts, published online ahead of print on 1 July 2009 J. Clin. Microbiol. doi: /jcm Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Rapid and Simple Determination of Ciprofloxacin Resistance in Clinical Strains of Escherichia coli Rebeca Santiso a b, María Tamayo a b, José Luis Fernández a b *, Mª del Carmen Fernández c, Francisca Molina c, Rosa Villanueva c, Jaime Gosálvez d, Germán Bou c * a INIBIC-Complejo Hospitalario Universitario Juan Canalejo, Unidad de Genética, As Xubias 84, A Coruña, Spain. b Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, Avda. de Montserrat s/n, A Coruña, Spain. c INIBIC-Complejo Hospitalario Universitario Juan Canalejo, Servicio de Microbiología, As Xubias, 84, A Coruña, Spain. d Unidad de Genética, Facultad de Biología, Universidad Autónoma de Madrid, Madrid, Spain. * Correspondence: Dr. José Luis Fernández Sección de Genética y Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, As Xubias, 84, A Coruña, Spain Tel: Fax: JLFernandez@canalejo.org ; joseluis.fernandez@cog.es Dr. Germán Bou Laboratorio de Microbiología Complejo Hospitalario Universitario Juan Canalejo Xubias s/n, 3ª Planta Ed. Sur A Coruña, SPAIN Phone Fax germanbou@canalejo.org 1

2 ABSTRACT We recently reported a simple new in situ diffusion assay, developed as a kit, to visualize DNA fragmentation in single bacterial cells. Use of this assay in a collection of 95 genetically-unrelated Escherichia coli clinical strains resulted in correct identification of all of the isolates as resistant or susceptible to ciprofloxacin, consistent with the MIC results. This relevant information is obtained in 80 minutes. 2

3 MANUSCRIPT Fluoroquinolones are among the most frequently prescribed antimicrobial agents worldwide, with ciprofloxacin (CIP) being one of the most widely used. The activity of these agents is mediated by the production of DNA fragmentation through trapping of DNA gyrase and/or topoisomerase IV on bacterial DNA (6). Fluoroquinolone MICs are mainly increased by mutations in the genes encoding the protein targets DNA gyrase (gyra and gyrb) and topoisomerase IV (parc and pare), but also by increasing the levels of specific efflux pumps, such as AcrAB-TolC, or by the presence of plasmidmediated mechanisms, such as QnrA, QnrB, QnR, or the recently described aminoglycoside resistance gene AAC (6 )-Ib-cr (8-11). Escherichia coli causes almost 50% of community-acquired urinary infections. Treatment with fluoroquinolones is recommended in such cases, especially in areas with predominant resistance to trimethoprim/sulphamethoxazole. However, over 22% of clinical isolates of E. coli may be resistant to CIP and this tendency appears to be increasing (5, 14). Resistance to CIP in clinical isolates of E. coli obtained from blood cultures has reached 19% in Spain. In a recent study, 43% of E. coli isolates from the normal intestinal microflora of humans were found to be resistant to quinolones (2). Given the alarming progressive increase in antibiotic resistance worldwide, it is of great importance to identify the resistant bacteria as soon as possible, to avoid treatment failure and the spread of resistant microorganisms through misuse of antibiotics. Our research group recently developed a diffusion-based assay, developed as a kit, which enables the straightforward and rapid (45 min) discrimination of bacteria with fragmented DNA. The kit can be used to evaluate DNA damage induced by antimicrobial agents. Since the activity of quinolones such as CIP depends on the production of fragmentation of bacterial DNA, we investigated the potential usefulness 3

4 of the kit as a simple, fast procedure for detecting resistance to CIP in clinical situations by processing a collection of clinical strains of E. coli isolated from patients attending the Juan Canalejo Hospital. Bacterial strains, antibiotic susceptibility testing and growth conditions. The procedure for determining chromosomal DNA fragmentation in situ was first assayed in control E. coli strains harbouring known mechanisms of quinolone-resistance, as well as in 95 E. coli clinical strains isolated from outpatients (only one strain per patient was selected for further studies) attending the hospital in and displaying different degrees of susceptibility to CIP. The E. coli isolates were routinely grown in Luria Bertani (LB) broth (1% Bacto Tryptone, 0.5% yeast extract, 0.5% NaCl) or on LB agar at 37ºC in aerobic conditions. The minimum inhibitory concentrations (MICs) of ciprofloxacin were determined by automated microdilution (MicroScan Walkaway, Dade) and confirmed by Etest (ABBiodisk, Solna Sweden) according to the manufacturer s instructions. REP-PCR. For molecular typing of the clinical strains of E. coli a reaction with the primer pair REP1 5 - IIIGCGCCGICATCAGGC-3 and REP2 5 - ACGTCTTATCAGGCCTAC-3 was used to amplify putative REP-like elements in the genomic bacterial DNA, as previously reported (1). Strains belonging to the same DNA group showed identical profiles or highly similar profiles (up to two bands different). Determination of DNA fragmentation. The Micro-Halomax kit for fluorescence microscopy (Halotech DNA SL, Madrid, Spain) was used to detect DNA fragmentation cell by cell, as previously reported (7). The clinical strains of E. coli were grown overnight on LB agar plates and then resuspended in LB broth at an OD 600 of Five ml of culture were then incubated with or without two different concentrations (0.1 and 1 µg/ml) of CIP (Sigma Aldrich, Madrid) for 40 min at 37ºC. This was carried out as a 4

5 blind procedure, without prior knowledge of the MICs. The two different concentrations of CIP were used for the following reasons. At 0.1 µg/ml those bacterial isolates harbouring some quinolone-resistant mechanism (which may act as a first step to yielding high CIP resistance through a second mechanism) were able to be detected at this low concentration. Although they remain susceptible to CIP according to CLSI criteria (3), the clinical consequences of this altered CIP susceptibility remains unknown. On the other hand a bacterial isolate with a CIP MIC higher than 1 µg/ml is considered as intermediate (non-susceptible), according to CLSI criteria [CIP breakpoints were 1,2, 4 (S,I,R)], and fully resistant according to EUCAST criteria [CIP breakpoints were 0.5,>1 (S,R)] ( Briefly, after CIP incubation, the cells were immersed in an agarose microgel on a slide, lysed and stained with a fluorochrome, according to the manufacturer s instructions. CIP-induced DNA fragmentation is visualized as DNA spots that diffuse peripherally from the nucleoid. The greater the DNA fragmentation, the greater the number of DNA spots and the width of the diffusion zone around the residual nucleoid. The response to CIP-induced damage is quantified simply by measuring the width of the diffusion zone of DNA fragments emerging from the border of the nucleoid. Five different categories of DNA fragmentation were determined, according to the width of the diffusion zone: grade 0: undamaged; grade I: low level of damage; grade II: intermediate damage; grade III: high level of damage; and grade IV: massive fragmentation (12). Control strains. The above mentioned conditions were tested with three control strains of E. coli harbouring specific mechanisms involved in reduced susceptibility to CIP (Figure). An E. coli strain J53 (CIP MIC of µg/ml) showed massive fragmentation at 0.1 and 1 µg/ml of CIP (grade IV at either concentration); the same E. 5

6 coli J53 harbouring the previously reported plasmid-mediated quinolone resistant determinant (9) (qnra1) (CIP MIC of 0.19 µg/ml) showed a high level of fragmentation at 1 µg/ml (grade III) but no effect at 0.1 µg/ml of CIP. A very similar result was obtained with strain C-15 (CIP MIC of 0.25 µg/ml), which displayed an S83L replacement at GyrA (13). Nevertheless, the effect of 1 µg/ml CIP was much lower than in strain J53 qnra1, and only grade II fragmentation was achieved. Otherwise, the E. coli clinical isolate C1341 (4) harbouring replacements at GyrA (S83L and D87Y) and ParC (S80I) (CIP MIC of 4 µg/ml) showed no DNA fragmentation at either concentration of CIP, thus revealing the ability of the method to detect resistance to CIP and also subtle differences in the degree of susceptibility to CIP. Results and discussion. A total of 110 clinical strains of E. coli were initially selected. However, 15 out of 110 isolates were discounted due to a possible genetic relationship, as revealed by REP-PCR. Therefore 95 isolates were subjected to further study of DNA fragmentation. According to the MIC obtained by microdilution and CLSI criteria, 74 isolates were categorized as resistant to CIP (77.9%) and 21 were categorized as susceptible to CIP (22.1%). The DNA fragmentation assay correctly identified all 74 CIP-resistant strains. As regards the susceptible strains all bacterial isolates were correctly assigned as susceptible to CIP (100% sensitivity and specificity). Moreover, in two cases the DNA fragmentation assay was able to detect bacterial strains with a CIP MIC of 1 µg/ml, grade II-III of DNA damage at 1 µg/ml, but no damage (grade 0) at the lower concentration of CIP. Although these bacterial isolates are considered fully susceptible to CIP following CLSI criteria, they appeared to harbour some mechanism that operates at a low level, which may lead -in a later step- to a second mutation/mechanism and thus to high CIP resistance. The clinical impact of these strains with low level of 6

7 resistance to quinolones (or CIP) remains unknown. However, microbiology laboratories should report and alert clinicians to the presence of these isolates with reduced susceptibility to CIP. The present findings are important in the field of antimicrobial resistance as results can be obtained in 80 minutes (including the 40 min of CIP incubation) as regards the susceptibility or resistance to CIP of an E. coli clinical strain, as compared with the 16 to 20 h needed to read a MIC to CIP by either microdilution or E-test. Preliminary results with five E. coli strains fully susceptible to CIP showed that when they were growing exponentially in LB broth culture rather than on LB agar, CIP sensitivity was immediately detected, without the need for prior incubation for 40 min. The presence of CIP during the 8 min required to prepare the microgel by mixing the aliquot of the culture with CIP and then with the liquid agarose following by cooling at 4ºC in the freezer, was sufficient to visualize CIP-induced DNA fragmentation. In summary, the procedure (as a kit) reported here is a simple, fast, reproducible and accurate method that enables routine determination of the susceptibility or resistance of clinical isolates of E. coli to CIP. Such rapid assessment may be of great interest to avoid treatment failure in urinary tract infections and particularly in life-threatening infections such as bacteraemia, frequently caused by E. coli isolates. Further experiments are in progress to implement the technology in other combinations of antimicrobial agents /microorganisms of high clinical impact, in order to detect multiresistant microorganisms in less than two hours. This study was supported by the Spanish Network for Research on Infectious Diseases- REIPI-(Instituto de Salud Carlos III, RD06/0008/0025), Fondo de Investigaciones 7

8 Sanitarias (PI061398, PI081368) and SERGAS (PS07/90) and grants from Xunta de Galicia (07CSA050916PR and INCITE07PXI916201ES). We also thank Dr L. Martínez, Dr J. Vila and Dr C. Torres for the kind gift of control strains of E. coli. REFERENCES 1. Bou G., M. Cartelle, M. Tomas, D. Canle, F. Molina, R. Moure, JM. Eiros, and A. Guerrero Identification and broad dissemination of the CTX-M-14 beta-lactamase in different Escherichia coli strains in the northwest area of Spain. J. Clin. Microbiol. 40: Chu YW., TK. Cheung, CH. Wong, GK. Tsang, K. Lee,SS. Lau, KM. Kam Quinolone resistance and correlation to other antimicrobial resistances in faecal isolates of Escherichia coli in Hong Kong. Chemotherapy. 54: Clinical and Laboratory Standars Institute Performance standards for antimicrobial susceptibility testing. Approved M100-S16. Vol. 26, No.1.Wayne, Pa. 4. Costa D., P.Poeta, Y. Saenz, L. Vinué, AC. Coelho, M. Matos, B. Rojo- Bezares, J. Rodrigues, C. Torres Mechanisms of antibiotic resistance in Escherichia coli isolates recovered from wild animals. Microbial Drug Resistance.14: Daza R., J. Gutierrez, G. Piedrola Antibiotic susceptibility of bacterial strains isolated from patients with community-acquired urinary tract infections. Int. J. Antimicrob Agents.18:

9 6. Drlica K., M. Malik, RJ. Kerns, X. Zhao Quinolone-mediated bacterial death. Antimicrob. Agents Chemother. 52: Fernández JL., M. cartelle, L. Muriel, R. Santiso, M. Tamayo, V. Goyanes, J. Gosálvez, and G. Bou DNA fragmentation in microorganisms assessed in situ. Appl. Environ. Microbiol. 74: Jacoby G.,V. Cattoir, D. Hooper, L. Martıinez-Martıinez, P. Nordmann,, A. Pascual, L. Poirel, and M. Wang qnr Gene Nomenclature. Antimicrob. Agents Chemother. 52: Martínez-Martínez M, Pascual A, Jacoby GA Quinolone resistance from a transferable plasmid. Lancet. 351: Robicsek A, J. Strahilevitz J, GA. Jacoby, M. Macielag, D. Abbanat, CH. Park, K. Bush, and DC. Hooper Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat. Med. 12 : Ruiz J. Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. J. Antimicrob. Chemother : Tamayo M., R. Santiso, J. Gosalvez, G. Bou and JL. Fernández Rapid assessment of ciprofloxacin effect on chromosomal DNA from Escherichia coli with an in situ DNA fragmentation assay. BMC Microbiol. In press. 13. Vila J., J. Ruiz, P. Goñi, MT. Jimenez de Anta Detection of mutations in parc in quinolone-resistant clinical isolates of Escherichia coli. Antimicrob. Agents Chemother. 40:

10 14. Warren JW., E. Abrutyn, JR. Hebel, JR. Johnson, AJ. Schaeffer, WE. Stamm Guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women. Infectious Diseases Society of America (IDSA). Clin. Infect. Dis. 29:

11 FIGURE LEGENDS Figure. Representative images of four strains of E. coli incubated with CIP for 40 min at doses of 0 µg/ml (a-d), 0.1 µg/ml (e-h) and 1 µg/ml (i-l), then processed with the kit to determine chromosomal DNA fragmentation, and stained with SYBR Gold. From left to right: first line (a, e, i), strain J53 (MIC of µg/ml); second line (b, f, j), strain J53qnrA1 (MIC of 0.19 µg/ml); third line (c, g, k), strain C-15 strain (MIC of 0.25 µg/ml), and the last line (d, h, l), strain C1341 (MIC of 4 µg/ml). The nucleoids from the fully sensitive strain J53 appear massively fragmented by 0.1 (e) and 1 (i) µg/ml CIP. When this strain harboured the qnra1 plasmid, 0.1 µg/ml CIP did not cause DNA fragmentation (f) and only 1 µg/ml CIP produced a high level of DNA fragmentation (j). The same occurred with strain C-15, although the level of DNA fragmentation by 1 µg/ml CIP was lower than in strain J53qnrA1 (k). With strain C1341, neither 0.1 µg/ml (h) nor 1 µg/ml (l) caused DNA fragmentation. 11

12 Figure 12