388
Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 383–393
Table 1 Effect of glutathione (GSH) on the minimum inhibitory concentration (MIC) of different quinolone derivatives in Escherichia coli K-12 strain MG165 Antibiotic
MIC (g/mL) Without GSH
With GSH
Inhibition of antibacterial action by GSH
Chemical groups at C-6 and C-7 position in quinolone molecule 6-H 7-H 6,7-Heterocyclic ring 6,7-Heterocyclic ring 6-F 7-H 6-H 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-[(3R,5S) 3,5-dimethylpiperazin-1-yl] 6-F 7-diazabicyclo 6-F 7-[(4Z)-3-(aminomethyl)-4-methoxyimino-pyrrolidin-1 yl]
Nalidixic acid
4
4
No
Oxolinic acid Cinoxacin Flumequine
1 4 0.8
1 4 0.8
No No No
Pipemidic acid
4
Norfloxacin
0.001
0.1
Yes
Ciprofloxacin
0.03
0.3
Yes
Ofloxacin
0.05
>1.0
Yes
Pefloxacin
0.01
1.0
Yes
Lomefloxacin
0.001
0.01
Yes
Gatifloxacin
0.001
0.01
Yes
Sparfloxacin
25 >25 >25 >25 >25 >25
– – – – – – – – – – – – – – – – – – – – N4S – – – – – –
No No No N/A Amoxicillin and gentamicin for 2 weeks No No No Amoxicillin and gentamicin for 2 weeks No SXT for 1 year No N/A N/A Yes No N/A N/A SXT for 1 year No No No N/A No N/A No No
Pneumonia CWD CWD CWD Endocarditis Arthritis CWD Arthritis Endocarditis Arthritis CWD (relapse)c CWD N/A CWD CWD CWD CWD Endocarditis CWD (relapse)c CWD Arthritis CWD CWD Endocarditis CWD CWD CWD
MIC, minimum inhibitory concentration; ATB, antibiotic treatment; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; CWD, Classic Whipple’s disease; N/A, not available; SXT, trimethoprim/sulfamethoxazole. a –, no mutation; N4S, presence of an N4S mutation with a corresponding amino acid change of asparagine to serine. b Strains were previously shown to be susceptible to sulphonamides in vitro [2]. c CWD with neurological relapse.
in 12-well plates. The antibiotic was diluted in culture medium at concentrations of 1, 10 and 25 g/mL. Antibiotic-free wells were used as growth controls. During the experiment, cultures were harvested and compared at Days 0 and 10. Growth was evaluated by counting bacteria by flow cytometry, and a strain was considered to be resistant if the size of the initial inoculum increased by a factor of ≥3 times. Briefly, the number of bacteria in each well was counted using an AccuriTM C6 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ). The accuracy of flow cytometry to detect T. whipplei in the axenic medium was tested prior to the experiment using known serial dilutions of the bacterium in its growing medium. The folP gene was also amplified and sequenced for all isolates as previously reported [3]. In all cases, T. whipplei grew more rapidly in cultures without antibiotics. However, significantly more bacterial growth (≥3-fold increase in the number of bacteria) was noticed at 10 days after incubation with antibiotics to which T. whipplei was hypothesised to be resistant. Of the 27 strains, 16 (59.3%) were found to be resistant to sulfadiazine at 1 g/mL, 11 (40.7%) at 10 g/mL and 7 (25.9%) at 25 g/mL (Table 1). The minimum inhibitory concentration breakpoint for sulfadiazine is ≥10 g/mL [4]. One strain presented an N4S mutation at the folP locus, which is known to predict secondary sulphonamide failure [3]. One patient with a clinically resistant strain (Neuro 1) had been treated previously with a 1-year course of SXT but relapsed 2 years after treatment. The results of the current study suggest that many strains of T. whipplei are resistant to sulphonamides. Therefore, we argue that SXT cannot be used as a standard treatment for Whipple’s disease because all strains of T. whipplei are naturally resistant to trimethoprim and many of our tested strains are resistant to sulphonamides. Treatments comprising several antibiotics including SXT may have been efficient due to the other drugs prescribed
at the same time, leading to the opinion that SXT was usually efficient. These findings emphasise that culture techniques represent a critical step in the development of therapeutic strategies for infectious diseases. Our simplified method of antibiotic testing may be used as a standard test of disease resistance to antibiotics, including doxycycline, which is the reference treatment for any isolated strain of T. whipplei. We recommend that SXT is finally abandoned as a treatment for Whipple’s disease. Acknowledgments The authors thank Marion Le Bideau and Céline Cal for technical assistance. Funding: Institut Hospitalo-Universitaire Méditerranée Infection (Marseille, France). Competing interests: None declared. Ethical approval: Not required. References [1] Lagier JC, Fenollar F, Lepidi H, Giorgi R, Million M, Raoult D. Treatment of classic Whipple’s disease: from in vitro results to clinical outcome. J Antimicrob Chemother 2014;69:219–27. [2] Boulos A, Rolain JM, Mallet MN, Raoult D. Molecular evaluation of antibiotic susceptibility of Tropheryma whipplei in axenic medium. J Antimicrob Chemother 2005;55:178–81. [3] Bakkali N, Fenollar F, Biswas S, Rolain JM, Raoult D. Acquired resistance to trimethoprim–sulfamethoxazole during Whipple disease and expression of the causative target gene. J Infect Dis 2008;198:101–8. [4] Arreaza L, de La Fuente L, Vázquez JA. Antibiotic susceptibility patterns of Neisseria meningitidis isolates from patients and asymptomatic carriers. Antimicrob Agents Chemother 2000;44:1705–7. [5] Renesto P, Crapoulet N, Ogata H, La Scola B, Vestris G, Claverie JM, et al. Genomebased design of a cell-free culture medium for Tropheryma whipplei. Lancet 2003;362:447–9.
390
Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 383–393
Florence Fenollar a,b Céline Perreal a,b Didier Raoult a,b,∗ a Aix-Marseille Université, Unité des Rickettsies, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), CNRS-IRD-INSERM UMR 7278, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France b APHM, CHU Timone, Pôle Infectieux, 13005 Marseille, France ∗ Corresponding
author at: Aix-Marseille Université, Unité des Rickettsies, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), CNRS-IRD-INSERM UMR 7278, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. E-mail address: [email protected] (D. Raoult) 20 January 2014 http://dx.doi.org/10.1016/j.ijantimicag.2014.01.015
Prevalence of plasmid-mediated quinolone resistance determinants in extended-spectrum -lactamase-producing and -non-producing enterobacteria in Spain Sir, Plasmid-mediated quinolone resistance (PMQR) is mainly associated with Qnr proteins and aac(6 )-Ib-cr, with oqxAB and qepA genes being seldom described [1]. PMQR determinants frequently appear associated with extended-spectrum -lactamase (ESBL) genes [1]. We report the prevalence and mechanisms of PMQR in ESBL-producing and -non-producing enterobacteria clinical isolates obtained in Murcia (south-eastern Spain). In total, 312 consecutive enterobacteria clinical isolates were studied, including 155 Escherichia coli, 46 Klebsiella pneumoniae, 32 Proteus mirabilis, 23 Salmonella enterica, 19 Serratia marcescens, 18 Enterobacter cloacae, 8 Citrobacter freundii, 7 Klebsiella oxytoca, 2 Enterobacter aerogenes, 1 Citrobacter koseri and 1 Morganella morganii. Susceptibility testing and ESBL confirmation were performed
by conventional methods. ESBLs and PMQR genes (qnrA, qnrB, qnrC, qnrS, qepA and aac(6 )-Ib-cr) were assessed by PCR in all clinical isolates. ESBLs and qnr genes were characterised by DNA sequencing. Mutations in gyrA, gyrB, parC and parE genes were analysed by PCR and sequencing in PMQR-positive isolates. Clonality among PMQR-positive isolates was evaluated by repetitive sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE). Fisher’s Exact with mid-P method were used for statistical significance. A p-value of
Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 383–393
Table 1 Effect of glutathione (GSH) on the minimum inhibitory concentration (MIC) of different quinolone derivatives in Escherichia coli K-12 strain MG165 Antibiotic
MIC (g/mL) Without GSH
With GSH
Inhibition of antibacterial action by GSH
Chemical groups at C-6 and C-7 position in quinolone molecule 6-H 7-H 6,7-Heterocyclic ring 6,7-Heterocyclic ring 6-F 7-H 6-H 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-[(3R,5S) 3,5-dimethylpiperazin-1-yl] 6-F 7-diazabicyclo 6-F 7-[(4Z)-3-(aminomethyl)-4-methoxyimino-pyrrolidin-1 yl]
Nalidixic acid
4
4
No
Oxolinic acid Cinoxacin Flumequine
1 4 0.8
1 4 0.8
No No No
Pipemidic acid
4
Norfloxacin
0.001
0.1
Yes
Ciprofloxacin
0.03
0.3
Yes
Ofloxacin
0.05
>1.0
Yes
Pefloxacin
0.01
1.0
Yes
Lomefloxacin
0.001
0.01
Yes
Gatifloxacin
0.001
0.01
Yes
Sparfloxacin
25 >25 >25 >25 >25 >25
– – – – – – – – – – – – – – – – – – – – N4S – – – – – –
No No No N/A Amoxicillin and gentamicin for 2 weeks No No No Amoxicillin and gentamicin for 2 weeks No SXT for 1 year No N/A N/A Yes No N/A N/A SXT for 1 year No No No N/A No N/A No No
Pneumonia CWD CWD CWD Endocarditis Arthritis CWD Arthritis Endocarditis Arthritis CWD (relapse)c CWD N/A CWD CWD CWD CWD Endocarditis CWD (relapse)c CWD Arthritis CWD CWD Endocarditis CWD CWD CWD
MIC, minimum inhibitory concentration; ATB, antibiotic treatment; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; CWD, Classic Whipple’s disease; N/A, not available; SXT, trimethoprim/sulfamethoxazole. a –, no mutation; N4S, presence of an N4S mutation with a corresponding amino acid change of asparagine to serine. b Strains were previously shown to be susceptible to sulphonamides in vitro [2]. c CWD with neurological relapse.
in 12-well plates. The antibiotic was diluted in culture medium at concentrations of 1, 10 and 25 g/mL. Antibiotic-free wells were used as growth controls. During the experiment, cultures were harvested and compared at Days 0 and 10. Growth was evaluated by counting bacteria by flow cytometry, and a strain was considered to be resistant if the size of the initial inoculum increased by a factor of ≥3 times. Briefly, the number of bacteria in each well was counted using an AccuriTM C6 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ). The accuracy of flow cytometry to detect T. whipplei in the axenic medium was tested prior to the experiment using known serial dilutions of the bacterium in its growing medium. The folP gene was also amplified and sequenced for all isolates as previously reported [3]. In all cases, T. whipplei grew more rapidly in cultures without antibiotics. However, significantly more bacterial growth (≥3-fold increase in the number of bacteria) was noticed at 10 days after incubation with antibiotics to which T. whipplei was hypothesised to be resistant. Of the 27 strains, 16 (59.3%) were found to be resistant to sulfadiazine at 1 g/mL, 11 (40.7%) at 10 g/mL and 7 (25.9%) at 25 g/mL (Table 1). The minimum inhibitory concentration breakpoint for sulfadiazine is ≥10 g/mL [4]. One strain presented an N4S mutation at the folP locus, which is known to predict secondary sulphonamide failure [3]. One patient with a clinically resistant strain (Neuro 1) had been treated previously with a 1-year course of SXT but relapsed 2 years after treatment. The results of the current study suggest that many strains of T. whipplei are resistant to sulphonamides. Therefore, we argue that SXT cannot be used as a standard treatment for Whipple’s disease because all strains of T. whipplei are naturally resistant to trimethoprim and many of our tested strains are resistant to sulphonamides. Treatments comprising several antibiotics including SXT may have been efficient due to the other drugs prescribed
at the same time, leading to the opinion that SXT was usually efficient. These findings emphasise that culture techniques represent a critical step in the development of therapeutic strategies for infectious diseases. Our simplified method of antibiotic testing may be used as a standard test of disease resistance to antibiotics, including doxycycline, which is the reference treatment for any isolated strain of T. whipplei. We recommend that SXT is finally abandoned as a treatment for Whipple’s disease. Acknowledgments The authors thank Marion Le Bideau and Céline Cal for technical assistance. Funding: Institut Hospitalo-Universitaire Méditerranée Infection (Marseille, France). Competing interests: None declared. Ethical approval: Not required. References [1] Lagier JC, Fenollar F, Lepidi H, Giorgi R, Million M, Raoult D. Treatment of classic Whipple’s disease: from in vitro results to clinical outcome. J Antimicrob Chemother 2014;69:219–27. [2] Boulos A, Rolain JM, Mallet MN, Raoult D. Molecular evaluation of antibiotic susceptibility of Tropheryma whipplei in axenic medium. J Antimicrob Chemother 2005;55:178–81. [3] Bakkali N, Fenollar F, Biswas S, Rolain JM, Raoult D. Acquired resistance to trimethoprim–sulfamethoxazole during Whipple disease and expression of the causative target gene. J Infect Dis 2008;198:101–8. [4] Arreaza L, de La Fuente L, Vázquez JA. Antibiotic susceptibility patterns of Neisseria meningitidis isolates from patients and asymptomatic carriers. Antimicrob Agents Chemother 2000;44:1705–7. [5] Renesto P, Crapoulet N, Ogata H, La Scola B, Vestris G, Claverie JM, et al. Genomebased design of a cell-free culture medium for Tropheryma whipplei. Lancet 2003;362:447–9.
390
Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 383–393
Florence Fenollar a,b Céline Perreal a,b Didier Raoult a,b,∗ a Aix-Marseille Université, Unité des Rickettsies, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), CNRS-IRD-INSERM UMR 7278, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France b APHM, CHU Timone, Pôle Infectieux, 13005 Marseille, France ∗ Corresponding
author at: Aix-Marseille Université, Unité des Rickettsies, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), CNRS-IRD-INSERM UMR 7278, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. E-mail address: [email protected] (D. Raoult) 20 January 2014 http://dx.doi.org/10.1016/j.ijantimicag.2014.01.015
Prevalence of plasmid-mediated quinolone resistance determinants in extended-spectrum -lactamase-producing and -non-producing enterobacteria in Spain Sir, Plasmid-mediated quinolone resistance (PMQR) is mainly associated with Qnr proteins and aac(6 )-Ib-cr, with oqxAB and qepA genes being seldom described [1]. PMQR determinants frequently appear associated with extended-spectrum -lactamase (ESBL) genes [1]. We report the prevalence and mechanisms of PMQR in ESBL-producing and -non-producing enterobacteria clinical isolates obtained in Murcia (south-eastern Spain). In total, 312 consecutive enterobacteria clinical isolates were studied, including 155 Escherichia coli, 46 Klebsiella pneumoniae, 32 Proteus mirabilis, 23 Salmonella enterica, 19 Serratia marcescens, 18 Enterobacter cloacae, 8 Citrobacter freundii, 7 Klebsiella oxytoca, 2 Enterobacter aerogenes, 1 Citrobacter koseri and 1 Morganella morganii. Susceptibility testing and ESBL confirmation were performed
by conventional methods. ESBLs and PMQR genes (qnrA, qnrB, qnrC, qnrS, qepA and aac(6 )-Ib-cr) were assessed by PCR in all clinical isolates. ESBLs and qnr genes were characterised by DNA sequencing. Mutations in gyrA, gyrB, parC and parE genes were analysed by PCR and sequencing in PMQR-positive isolates. Clonality among PMQR-positive isolates was evaluated by repetitive sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE). Fisher’s Exact with mid-P method were used for statistical significance. A p-value of
