Moxifloxacin resistance is prevalent among Bacteroides and Prevotella species in Greece

Journal of Antimicrobial Chemotherapy (2008) 62, 137 141 doi:10.1093/jac/dkn134 Advance Access publication 1 April 2008 Moxifloxacin resistance is prevalent among Bacteroides and Prevotella species in Greece Joseph Papaparaskevas 1 *, Angeliki Pantazatou 2, Anastasia Katsandri 1,2, Dimitra P. Houhoula 1, Nicholas J. Legakis 1, Athanassios Tsakris 1 and Athina Avlamis 2 1 Department of Microbiology, Medical School, University of Athens, 11527 Athens, Greece; 2 Department of Microbiology, Laikon University Hospital, 11527 Athens, Greece Received 29 November 2007; returned 2 February 2008; revised 14 February 2008; accepted 5 March 2008 Objectives: Moxifloxacin is recommended in the empirical treatment of infections involving Gram-negative anaerobes. However, current European data regarding its activity against anaerobic pathogens are limited. In order to evaluate its potency, we comparatively studied the activity of moxifloxacin against recently isolated Gram-negative anaerobes. Methods: Four hundred and ninety-five Gram-negative anaerobic clinical isolates (296 Bacteroides fragilis group, 58 non-fragilis Bacteroides spp. and 141 Prevotella spp.) were prospectively recovered in six Greek hospitals. Moxifloxacin MICs were determined in comparison with those of penicillin, piperacillin/tazobactam, cefoxitin, imipenem, metronidazole and clindamycin. Results: Overall moxifloxacin MIC 50 and MIC 90 were 2 and 32 mg/l, respectively. Based on the current CLSI breakpoints (susceptible, 2 mg/l; resistant, 8 mg/l), almost half of the total isolates (49%) were non-susceptible to moxifloxacin (32% resistant; 17% intermediate). This was more evident among the non-fragilis Bacteroides species, where 47% of the isolates were resistant and 14% intermediate to moxifloxacin. Species variation was noticed, with the highest non-susceptible rates detected among Prevotella oralis (90%), Prevotella bivia (80%), Bacteroides thetaiotaomicron (75%), Bacteroides uniformis (70%) and Bacteroides capillosus (67%) species. Among the 19 (4%) isolates that were metronidazole non-susceptible (MIC 16 mg/l), only 4 (21%) were additionally non-susceptible to moxifloxacin. Conclusions: High resistance rates to moxifloxacin among Bacteroides and Prevotella spp. were recorded, exceeding those previously reported in Europe and contraindicating its use as monotherapy for infections involving Gram-negative anaerobes without prior microbiological confirmation. For empirical usage, moxifloxacin should be combined with metronidazole in order to cover for these pathogens. Keywords: metronidazole, susceptibility, anaerobes, epidemiology, Bacteroides spp., Prevotella spp. Introduction Although anaerobic bacteria play a significant role in polymicrobial infections, 1 not all hospital laboratories include the performance of anaerobic cultures in their routine protocols, and only selected centres perform susceptibility testing. In that respect, antimicrobial chemotherapy is mainly empirical and is based on data from multicentre studies. 1,2 Nevertheless, resistance to all antimicrobial agents with activity against anaerobes has been reported, as well as great variations in different geographical areas, 1 thus demanding more frequent periodic resistance surveillance. Intra-abdominal infections are commonly due to mixed aerobic and anaerobic bacteria requiring broad-spectrum antibiotic therapy. 2 Moxifloxacin is a relatively new methoxyquinolone with potent antimicrobial activity against both aerobic and anaerobic bacteria. 3 In that respect, it has been included in the guidelines for treatment of intra-abdominal infections. 2 However, a limited number of recent studies, mainly from the USA, indicate an increase in moxifloxacin resistant rates among Bacteroides and Prevotella spp. 4,5 Comparable recent European data are scarce and only a single study reported a shift towards increased resistance. 6... *Corresponding author. Tel: þ30-210-7462142; Fax: þ30-210-7462143; E-mail: ipapapar@med.uoa.gr... 137 # The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Papaparaskevas et al. The present study was undertaken to determine the comparative in vitro activities of moxifloxacin against Bacteroides and Prevotella spp. clinical isolates, in order to verify its usefulness in the empirical treatment of multibacterial infections involving Gram-negative anaerobes. Materials and methods Gram-negative anaerobic clinical isolates belonging to Bacteroides or Prevotella genus were consecutively collected during 2006 07, from six general hospitals in Athens (five hospitals with adult patients and one with paediatric), with 3000 beds in total. All isolates were non-duplicate and derived from documented infections, for which patients received antimicrobial chemotherapy. The initial bacterial isolation and identification was performed in the individual laboratories. Subsequently, all isolates were collected from the Laboratory of Laikon University Hospital, where level III species identification was confirmed using the special potency disc method, grown in the presence of 20% bile (Bacteroides-Bile Esculin agar plates; Bioprepare, 16346, Gerakas, Greece), indole reaction (Bioprepare), and the biochemical profile using the ID32ANA system (biomérieux, Marcy l Étoile, France). 7 MICs of penicillin, piperacillin/tazobactam, cefoxitin, imipenem, metronidazole, clindamycin and moxifloxacin were determined by the Etest method (AB Biodisk, Solna, Sweden), according to the manufacturer s instructions. Briefly, a suspension with a turbidity equivalent to that of a 1.0 McFarland standard in brucella broth was prepared from a 48 h culture plate and spread onto brucella agar plates supplemented with 5% (v/v) horse blood, vitamin K 1 (1 mg/l) and haemin (5 mg/l). Susceptibility testing plates were incubated at 378C for 48 h in a Bactron 1.5 Anaerobic Chamber (Cheldon Manufacturing, Cornelius, OR, USA). Susceptibilities were interpreted according to the CLSI guidelines. 8 For moxifloxacin, the recent CLSI breakpoints were used (susceptible, 2 mg/l; intermediate, 4 mg/l; resistant, 8mg/L). 8 Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741 strains were used for quality control. 8 Throughout the study, anaerobiosis was ensured with methylene blue strips and resazurine. Results A total of 495 non-repetitive Gram-negative anaerobic bacteria, identified as Bacteroides or Prevotella spp., were isolated during the period of the study. Abdominal infections (55.3%) along with diabetic foot and other soft tissue infections (24.6%) were the most common sources of isolation; less frequent sources were respiratory tract infections (11.2%) and bacteraemias (8.9%). The dominant species was B. fragilis (n ¼ 165; 33.3%) and the majority of the isolates belonged to B. fragilis group (n ¼ 296, 59.8%), whereas 58 isolates (11.7%) were identified as non-fragilis Bacteroides spp. and 141 isolates (28.5%) were identified as Prevotella spp. MIC ranges, MIC 50 and MIC 90 values, and rates of resistant, intermediate and susceptible isolates for each antimicrobial agent among all Gram-negative bacteria, as well as B. fragilis group, Bacteroides spp. non-fragilis, Prevotella spp. and the most frequent species, are summarized in Table 1. Overall moxifloxacin MIC 50 and MIC 90 were 2 and 32 mg/l, respectively. Almost half of the total isolates (49%) were nonsusceptible to moxifloxacin (32% resistant; 17% intermediate). This was more evident among non-fragilis Bacteroides spp., where 47% of the isolates were resistant and 14% intermediate, whereas lower non-susceptible rates were observed for B. fragilis group (29% resistant; 13% intermediate) and Prevotella spp. (38% resistant; 4% intermediate). The high non-susceptible rate of the non-fragilis group should be attributed to the Bacteroides capillosus, which was the most prevalent species of this group and exhibited the highest non-susceptible rates (54% resistant; 13% intermediate). This is reported for the first time, as no other comparable data are available in the literature. Variations in the moxifloxacin resistance rates were recorded among the B. fragilis group species. Regarding the five most prevalent species (comprising 91% of the group isolates), B. thetaiotaomicron exhibited the highest non-susceptible rates (75%, all fully resistant), followed by Bacteroides uniformis (55% resistant, 15% intermediate), whereas lower non-susceptible rates were observed for Bacteroides distasonis (29% resistant, 11% intermediate), B. fragilis (16% resistant, 14% intermediate) and Bacteroides vulgatus (14% resistant, 16% intermediate). Variations were also recorded within the Prevotella spp. group, where the most prevalent species exhibited the highest resistance rates (Prevotella bivia, 71% resistant and 9% intermediate; Prevotella oralis, 62% resistant and 28% intermediate), whereas the rest of Prevotella spp. isolates exhibited an overall nonsusceptible rate of 29%. Regarding the remaining tested antimicrobials, 90% of the isolates were non-susceptible to penicillin (MIC, 1 mg/l), 34% to clindamycin (MIC, 4 mg/l), 26% to cefoxitin (MIC, 32 mg/l), 4% to metronidazole (MIC, 16 mg/l), 2% to piperacillin/tazobactam (MIC, 64 mg/l) and,1% to imipenem (MIC, 8 mg/l). Discussion Our study showed that resistance to moxifloxacin is prevalent among Gram-negative anaerobic species, and the resistance rates described herein are even higher than those reported previously. 4,5 Comparable recent data from Europe do not exist, apart from a Belgian study indicating a possible equivalent increase. 6 It should be noted that in two previous European studies for the period 1997 2002, 1,9 lower resistance rates to moxifloxacin were reported (16% to 19%, at a breakpoint of 8 mg/l), and only in certain Bacteroides species, an increasing trend was noted. 9 However, two recent studies from the USA, 10,11 one of which also examined the activity of moxifloxacin against Bacteroides spp. strains isolated before its introduction in clinical practice, 10 have shown increasing resistance rates over time to moxifloxacin (overall 28% to 34.5%, at a breakpoint of 8 mg/l), as well as other newer quinolones. This kind of cross-resistance may be attributed to previous heavy clinical use of fluoroquinolones, resulting possibly in mutations at the same gene target and explaining the geographical variations of resistance. 6,10 In comparison to other European countries, Greece has experienced higher quinolone consumption rates, 12 which may explain to a certain degree the findings of the present study. Given the fact, however, that phenotypic resistance to moxifloxacin is expected to emerge at a slower rate than in older fluoroquinolones, because it requires two mutations in gyra gene, 10 other mechanisms, like multidrug efflux pumps, 13 might also be responsible. 138

Moxifloxacin resistance among anaerobic bacteria Table 1. Activities of moxifloxacin and comparator agents against Bacteroides and Prevotella spp. clinical isolates MIC (mg/l) Susceptibility (%) a Species (no. of strains) Antimicrobial agent range MIC 50 MIC 90 R I S Total Gram-negatives (495) moxifloxacin 0.125 to.32 2.32 32 17 51 penicillin 0.016 to.256 64.256 88 2 10 piperacillin/tazobactam 0.016 to.256 0.25 8 1 1 98 cefoxitin 0.016 to.256 8 64 12 14 74 imipenem 0.016 to.32 0.064 0.5,1 0 99 clindamycin 0.016 to.256 1.256 27 7 66 metronidazole 0.016 to.32 0.5 1 3 1 96 Bacteroides spp. (354) moxifloxacin 0.125 to.32 2.32 32 16 52 penicillin 0.016 to.256 64.256 96 1 3 piperacillin/tazobactam 0.016 to.256 0.5 8 2 1 97 cefoxitin 0.125 to.256 16 64 15 17 68 imipenem 0.016 to.32 0.125 0.5,1 0 99 clindamycin 0.016 to.256 1.256 27 8 65 metronidazole 0.016 to.32 0.5 1 1 1 98 B. fragilis group b (296) moxifloxacin 0.125 to.32 2.32 29 13 58 penicillin 0.125 to.256 64.256 97 1 2 piperacillin/tazobactam 0.016 to.256 0.5 8 2 0 98 cefoxitin 0.125 to.256 16 128 17 15 68 imipenem 0.016 to.32 0.016.32,1 0 99 clindamycin 0.016 to.256 1.256 27 8 65 metronidazole 0.032 to.32 0.5 1 1 0 99 B. fragilis (165) moxifloxacin 0.125 to.32 2.32 16 14 70 penicillin 0.25 to.256 64.256 98 1 1 piperacillin/tazobactam 0.016 to.256 0.5 8 3 0 97 cefoxitin 0.5 to.256 16 64 12 13 75 imipenem 0.016 to.32 0.064 0.25,1 0 99 clindamycin 0.016 to.256 1 64 14 10 76 metronidazole 0.064 16 0.5 1 0 1 99 B. thetaiotaomicron (42) moxifloxacin 0.125 to.32 8.32 75 0 25 penicillin 0.125 to.256 64.256 98 1 1 piperacillin/tazobactam 0.064 32 1 16 0 0 100 cefoxitin 2 to.256 32 64 23 33 44 imipenem 0.016 2 0.125 0.25 0 0 100 clindamycin 0.016 to.256 2.256 30 14 56 metronidazole 0.125 1 0.5 1 0 0 100 B. uniformis (27) moxifloxacin 0.25 to.32 2.32 55 15 30 penicillin 0.5 to.256 64 128 98 1 1 piperacillin/tazobactam 0.016 8 0.5 4 0 0 100 cefoxitin 0.125 128 4 64 18 6 76 imipenem 0.016 2 0.125 1 0 0 100 clindamycin 0.064 to.256 1.256 35 12 53 metronidazole 0.125 1 0.5 0.5 0 0 100 B. distasonis (20) moxifloxacin 0.5 to.32 2.32 29 11 60 penicillin 16 to.256 64.256 100 0 0 piperacillin/tazobactam 0.032 32 2 4 0 0 100 cefoxitin 1 to.256 16 128 29 21 50 imipenem 0.032 2 0.25 1 0 0 100 clindamycin 0.032 to.256 4.256 43 14 43 metronidazole 0.032 4 0.25 1 0 0 100 B. vulgatus (15) moxifloxacin 0.125 to.32 1.32 14 16 70 penicillin 0.5 to.256 64.256 98 1 1 piperacillin/tazobactam 0.016 to.256 2 32 7 0 93 cefoxitin 0.25 to.256 4 32 7 13 80 Continued 139

Papaparaskevas et al. Table 1. Continued MIC (mg/l) Susceptibility (%) a Species (no. of strains) Antimicrobial agent range MIC 50 MIC 90 R I S imipenem 0.032 to.32 0.25 0.5 7 0 93 clindamycin 0.016 to.256 4.256 47 7 46 metronidazole 0.125 to.32 0.5 1 7 0 93 Bacteroides spp. non-fragilis c (58) moxifloxacin 0.125 to.32 4.32 47 14 39 penicillin 0.016 to.256 64.256 90 4 6 piperacillin/tazobactam 0.016 to.256 0.25 16 1 0 99 cefoxitin 0.25 128 8 32 9 23 68 imipenem 0.016 4 0.125 0.25 0 0 100 clindamycin 0.016 to.256 1.256 30 4 66 metronidazole 0.016 16 0.5 1 0 2 98 B. capillosus (44) moxifloxacin 0.125 to.32 8.32 54 13 33 penicillin 1 to.256 64.256 96 2 2 piperacillin/tazobactam 0.032 to.256 0.25 16 3 0 97 cefoxitin 0.25 32 8 32 12 16 72 imipenem 0.032 4 0.064 0.5 0 0 100 clindamycin 0.016 to.256 1 128 27 5 68 metronidazole 0.016 16 0.5 1 0 2 98 Prevotella spp. d (141) moxifloxacin 0.125 to.32 4 32 38 4 58 penicillin 0.016 to.256 4 128 69 2 29 piperacillin/tazobactam 0.016 4 0.016 0.5 0 0 100 cefoxitin 0.016 64 0.5 8 1 2 97 imipenem 0.016 1 0.032 0.125 0 0 100 clindamycin 0.016 to.256 0.064.256 31 2 67 metronidazole 0.016 to.32 0.25 2 8 0 92 P. bivia (35) moxifloxacin 0.5 to.32 8 32 71 9 20 penicillin 0.016 64 8 64 69 0 31 piperacillin/tazobactam 0.016 0.5 0.016 0.032 0 0 100 cefoxitin 0.032 2 0.5 1 0 0 100 imipenem 0.016 0.125 0.032 0.064 0 0 100 clindamycin 0.016 to.256 0.032 16 31 2 67 metronidazole 0.016 to.32 1 2 6 0 94 P. oralis (29) moxifloxacin 0.5 to.32 4 32 62 28 10 penicillin 0.016 to.256 8 128 70 2 28 piperacillin/tazobactam 0.016 2 0.125 1 0 0 100 cefoxitin 0.032 64 4 32 6 11 83 imipenem 0.016 1 0.064 0.25 0 0 100 clindamycin 0.016 to.256 1.256 33 6 61 metronidazole 0.032 to.32 0.5 2 8 0 92 a R, resistant; I, intermediate; S, susceptible. b Includes: B. fragilis (165), B. thetaiotaomicron (42), B. uniformis (27), B. distasonis (20), B. vulgatus (15), B. ovatus (9), B. merdae (7), B. caccae (6), B. eggerthii (5). c Includes: B. capillosus (44), B. ureolyticus (14). d Includes: P. bivia (35), P. oralis (29), P. buccae (12), P. disiens (12), P. loescheii (11), P. buccalis (10), P. indermedia (10), P. denticola (9), P. melaninogenica (9), P. ruminicola (4). The variation in the resistance of various species reported herein seems to differ from other studies, where isolates identified as B. vulgatus, 1,10,11 Bacteroides ovatus 1,6,10 or B. distasonis 10 were among the most resistant species, indicating variations not only among species but also between different geographic regions, as has been suggested previously. 1,10,11 In addition, although previous studies have reported even higher moxifloxacin resistance among certain Bacteroides species (such as B. vulgatus), 1,11 our study indicated that this resistance seems to affect many species belonging not only to Bacteroides but also to Prevotella genus. A high intermediate rate (MIC ¼ 4 mg/l) was recorded in many species, an observation that needs continuous active surveillance. Resistance rates to other antimicrobials with activity against anaerobes, such as penicillin, cefoxitin, piperacillin/tazobactam, clindamycin, and imipenem, reported here were similar to those from the last Greek study 14 and comparable to previously reported multicentre European data. 1 In addition, metronidazole resistance 140

Moxifloxacin resistance among anaerobic bacteria rates were equally low (overall 3% resistant and 1% intermediate), although a shift towards isolation of metronidazole-resistant Bacteroides spp. was detected. In the previous Greek study, 14 metronidazole resistance was 3% and was almost exclusively detected among isolates identified as Prevotella spp., whereas in the present study, as many as 44% of the metronidazole-resistant isolates were Bacteroides spp. A total of 19 isolates were metronidazole non-susceptible (14 isolates resistant, MIC 32 mg/l; 5 isolates intermediate, MIC ¼ 16 mg/l), of which only 4 (21%) were additionally moxifloxacin resistant (MIC 8 mg/l). In that respect, moxifloxacin, although exhibiting high resistance rates, seems to be effective against isolates conferring resistance to metronidazole, thus making its combination with metronidazole still an acceptable solution for the anaerobic pathogens of intra-abdominal infections, according to guidelines. 3 In conclusion, this multicentre study in Greece showed that moxifloxacin exhibited high intermediate and fully resistant rates against Bacteroides and Prevotella species, the highest reported so far in Europe, thus making full species identification of all anaerobes isolated from clinical specimens mandatory, as well as performance of susceptibility testing, in order to justify its administration as monotherapy. For empirical usage, moxifloxacin should be combined with metronidazole in order to cover for these pathogens. Acknowledgements We are thankful to the following members of the Hellenic Study Group for Gram-Negative Anaerobic Bacteria for contributing isolates to this study: Dr E. Papafrangas and Dr M. Kanellopoulou (Sismanoglion General Hospital), Dr C. Koutsia-Karouzou and Dr D. Kairis (Asklepeion General Hospital), Dr C. Trikka-Graphakos (Thriassion General Hospital), Dr H. Malamou-Ladas and Dr G. Ganteris (G. Gennimatas General Hospital), and Dr A. Vogiatzi (Penteli s Children Hospital). Funding This study was co-funded by the European Social Fund and National Resources (grants EPEAEK II Pythagoras II and Kapodistrias). Transparency declarations None to declare. References 1. Hedberg M, Nord CE. Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe. Clin Microbiol Infect 2003; 9: 475 88. 2. Solomkin JS, Mazuski JE, Baron EJ et al. Guidelines for the selection of anti-infective agents for complicated intra-abdominal infections. Clin Infect Dis 2003; 37: 997 1005. 3. Ackermann G, Schaumann R, Pless B et al. Comparative activity of moxifloxacin in vitro against obligately anaerobic bacteria. Eur J Clin Microbiol Infect Dis 2000; 19: 228 32. 4. Citron DM, Goldstein EJC, Merriam CV et al. Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents. J Clin Microbiol 2007; 45: 2819 28. 5. Goldstein EJC, Citron DM, Warren YA et al. In vitro activity of moxifloxacin against 923 anaerobes isolated from human intra-abdominal infections. Antimicrob Agents Chemother 2006; 50: 148 55. 6. Wybo I, Pierard D, Verschraegen I et al. Third Belgian multicenter survey of antibiotic susceptibility of anaerobic bacteria. J Antimicrob Chemother 2007; 59: 132 9. 7. Jousimies-Somer HR, Summanen P, Citron DM et al. Wadsworth KTL Anaerobic Bacteriology Manual, 6th edn. Belmont, CA: Star Publishing Company, 2002; 81 132. 8. Clinical and Laboratory Standards Institute. Methods for Antimicrobial Testing of Anaerobic Bacteria Seventh Edition: Approved Standard M11-A7. CLSI, Wayne, PA, USA, 2007. 9. Betriu C, Rodríguez-Avial I, Gomez M et al. Changing patterns of fluoroquinolone resistance among Bacteroides fragilis group organisms over a 6-year period (1997 2002). Diagn Microbiol Infect Dis 2005; 53: 221 3. 10. Golan Y, McDermott LA, Jacobus NV et al. Emergence of fluoroquinolone resistance among Bacteroides species. J Antimicrob Chemother 2003; 52: 208 13. 11. Snydman DR, Jacobus NV, McDermott LA et al. National survey on the susceptibility of Bacteroides fragilis group: report and analysis of trends in the United States from 1997 to 2004. Antimicrob Agents Chemother 2007; 51: 1649 55. 12. Ferech M, Coenen S, Malhotra-Kumar S et al. European Surveillance of Antimicrobial Consumption (ESAC): outpatient quinolone use in Europe. J Antimicrob Chemother 2006; 58: 423 7. 13. Miyamae S, Ueda O, Yoshimura F et al. A MATE family multidrug efflux transporter pumps out fluoroquinolones in Bacteroides thetaiotaomicron. Antimicrob Agents Chemother 2001; 45: 3341 6. 14. Katsandri A, Avlamis A, Pantazatou A et al. In vitro activities of tigecycline against recently isolated Gram-negative anaerobic bacteria in Greece, including metronidazole-resistant strains. Diagn Microbiol Infect Dis 2006; 55: 231 6. 141