Antibiotic Resistance in Prevotella Species Isolated from Patients with Cystic ﬁbrosis

J Antimicrob Chemother 2013; 68: 2369–2374 doi:10.1093/jac/dkt191 Advance Access publication 21 May 2013

Antibiotic resistance in Prevotella species isolated from patients with cystic ﬁbrosis

Laura J. Sherrard1,2, Kathryn A. Graham1,2, Stef J. McGrath1,2, Leanne McIlreavey1,2, Joseph Hatch3,4, Marianne S. Muhlebach3, Matthew C. Wolfgang3,4, Deirdre F. Gilpin1,2, J. Stuart Elborn1,5, Thamarai Schneiders5 and Michael M. Tunney1,2*

1CF & Airways Microbiology Group, Queen’s University Belfast, Belfast, UK; 2School of Pharmacy, Queen’s University Belfast, Belfast, UK; 3Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 4Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 5Centre for Infection & Immunity, School of Medicine, Dentistry & Biomedical Science, Queen’s University Belfast, Belfast, UK

*Corresponding author. School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK. Tel: +44-(0)28-90972087; Fax: +44-(0)28-90247794; E-mail: [email protected]

Received 13 March 2013; returned 29 March 2013; revised 11 April 2013; accepted 12 April 2013

Objectives: To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF) and non- CFpatientsand analyse the impact of antibiotic prescribing in the preceding yearon resistance amongstCFisolates. Methods: The susceptibility of 80 CF Prevotella isolates to 12 antibiotics was compared with that of 50 Prevotella isolates from invasive infections in people who did not have CF and 27 Prevotella isolates from healthy controls. Results: All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam, with only four isolates resistant to metronidazole. However, resistance to amoxicillin, ceftazidime and tetracycline was apparent in all groups. Significant differences in clindamycin resistance (UK CF, 56%; UK invasive, 10%) and co-amoxiclav non-susceptibility (UK CF, 32%; UK invasive, 12%) were observed between UK CF and UK invasive isolates. The likelihood of non-susceptibility to clindamycin and co-amoxiclav in UK CF isolates was 5.5-fold and 2.5-fold higher relative to that in UK invasive isolates, respectively. Azithromycin MICs were also significantly higher for CF isolates (P,0.001), which was associated with current prescription of azithromycin. More than 50% of clinical isolates tested in this study were b-lactamase positive. Conclusions: This study profiles antibiotic susceptibility in Prevotella spp. in CFand demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated.

Keywords: anaerobic bacteria, antimicrobial susceptibility, b-lactamase production

Introduction members of the oral flora in patientswith CF, non-CF bronchiectasis and chronic obstructive pulmonary disease (COPD).9 Prevotella spp. are obligate anaerobic bacteria and common Exposure of other members of the CF microbiota, such as members of the healthy microbiota at various body sites, including Prevotella spp., to antibiotics used in the treatment of CF pulmon- 1,2 the oral cavity and the respiratory tract. Recently, Prevotella spp. ary infection may also increase their resistance to antimicrobials. have been detected as part of diverse polymicrobial communities Therefore, in this study, we determined the antimicrobial suscepti- 3 – 8 in respiratory samples from cystic fibrosis (CF) patients. bility of a large number of clinical Prevotella isolates cultured from Although their precise role in CF lung disease is not yet clear, they patients with CF and those without CF and analysed the impact of may be clinically significant pathogens and contribute to infection antibiotic prescribing on resistance amongst CF isolates. Of the 12 and inflammation in the CF lung. antibiotics selected, 9 (amoxicillin, co-amoxiclav, azithromycin, Antibiotics are employed extensively to treat CF pulmonary in- ceftazidime, chloramphenicol, clindamycin, doxycycline, merope- fection and several studies have examined the effect of persistent nem and piperacillin/tazobactam) are used in the treatment of antibiotic use on bacterial resistance. For example, long-term CF lung infection and have putative activity against anaerobes. macrolide exposure has been shown to significantly increase One antibiotic (tobramycin) is used to treat CF lung infection, macrolide resistance amongst both respiratory pathogens and but has no putative anaerobic activity, and two antibiotics

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(metronidazole and tetracycline) have putative anaerobic activity, isolates had high MICs of tobramycin (≥64 mg/L). Resistance to but are not routinely used to treat CF lung infection. azithromycin was evident among the UK CF, UK invasive and US CF isolates (MIC90 .256 mg/L), but was less apparent in the UK healthy control isolates (MIC90 3 mg/L). All isolates tested were Methods susceptible to meropenem, piperacillin/tazobactam and chloram- Bacterial isolates and prior antibiotic use phenicol, with resistance to metronidazole (3%) rare. The clinical Prevotella isolates (n¼157) used in this study were split into four groups: (i) 57 isolates cultured from 25 adult (≥18 years) UK CF patients Comparison of antimicrobial susceptibility between UK CF, attending the CF clinic, Belfast, UK [cultured from sputum (n¼53) and invasive and healthy control isolates plaque (n¼4)]; (ii) 23 isolates cultured from 15 paediatric (,18 years) US CF patients attending the CF clinic, University of North Carolina, Chapel Comparison of the MICs for UK isolates (Table 1) showed significant Hill, NC, USA [cultured from bronchoalveolar lavage fluid (n¼14) and differences for ceftazidime (P¼0.035), clindamycin (P,0.001), sputum (n¼9)]; (iii) 50 isolates from invasive infections, including cultures co-amoxiclav (P,0.001), meropenem (P¼0.001), azithromycin from blood, abscesses and ulcers, from 50 UK non-CF patients, kindly pro- (P,0.001), chloramphenicol (P¼0.014) and doxycycline vided by Dr Valerie Hall (Anaerobe Reference Laboratory, Cardiff, UK); and (P¼0.020), with no differences evident for the remaining five anti- ≥ (iv) 27 isolates from 15 adult ( 18 years) UK healthy control subjects (cul- biotics. Post hoc tests revealed that CF isolates had significantly tured from induced sputum). Information on intravenous and oral antibio- higher MICs of clindamycin (P,0.001) and azithromycin tics prescribed in the year preceding culture of UK and US CF isolates was (P,0.001) compared with invasive and healthy control isolates. collected from patient hospital notes where available (Tables S1 and S2, Although there was no difference in either co-amoxiclav or doxy- available as Supplementary data at JAC Online). Quality assurance testing was performed using Bacteroides fragilis ATCC 25285, Pseudomonas cycline MICs between the CF and invasive isolates, both these aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Strepto- groups had significantly higher MICs of co-amoxiclav (CF, coccus pneumoniae ATCC 49619. P,0.001; invasive, P¼0.012) and doxycycline (CF, P¼0.027; invasive, P¼0.03) compared with healthy control isolates. For antibiotics with breakpoints approved by the CLSI, we deter- Antimicrobial susceptibility testing and b-lactamase mined the risk of antimicrobial non-susceptibility in UK CF isolates production relative to invasive isolates (Table 2). Significant differences in clin- The antimicrobial susceptibility of isolates was determined by Etest (AB damycin (P,0.001) and co-amoxiclav (P¼0.037) resistance were Biodisk, Hampshire, UK) in accordance with the manufacturer’s instruc- observed. The likelihood of non-susceptibility to clindamycin and tions. MICs were categorized as indicating resistance, intermediate resist- co-amoxiclavin CFisolateswas 5.5-fold and 2.5-fold higher relative ance or susceptibility according to the MIC breakpoints for anaerobes to that in invasive isolates, respectively. defined by the CLSI (Table 1). b-Lactamase production was determined using a nitrocefin assay as described previously.10 Current azithromycin prescription and antibiotic resistance in CF isolates Data analysis To investigate whether increased resistance to azithromycin was The susceptibility (MIC) of UK isolates in the three groups (CF, invasive and associated with current prescription of the antibiotic, isolates healthy control) to each antibiotic was compared using the Kruskal– ¼ Wallis test, with post hoc tests performed using the Mann–Whitney test were grouped [current prescription isolates (n 30) versus no with Bonferroni adjustment applied. The UK and US CF isolates were current prescription isolates (n¼32)], with current prescription grouped according to patient prescription of azithromycin (current prescrip- clearly associated with significantly higher azithromycin MICs tion versus no current prescription) and azithromycin MICs compared using (P¼0.005; Figure 1). Furthermore, isolatesfrom CFpatientsnot cur- the Mann–Whitney test. Spearman’s rank correlation coefficient was used rently prescribed azithromycin (n¼32) had significantly higher to measure the strength of the linear association between azithromycin azithromycin MICs (P¼0.011) compared with isolates (n¼17) MICs and clindamycin resistance. The CF isolates were also grouped accord- from UK healthy controls who were also not currently prescribed ing to patient prescription of b-lactam and tetracycline antibiotics in the azithromycin (Figure 1). There was also a positive correlation year prior to pulmonary sample collection (prescription versus no prescrip- between clindamycin resistance and high azithromycin MICs tion in the previous year) and b-lactam and tetracycline antibiotic MICs (r¼0.785, P,0.001). compared using the Mann–Whitney test. MICs were also compared in b-lactamase-positive versus b-lactamase-negative isolates using the Mann–Whitney test. b-Lactam/tetracycline prescription in the year preceding sample collection and antibiotic resistance Results Prescription of a b-lactam antibiotic (Tables S1 and S2) [prescription (n¼43) versus no prescription (n¼27)] was associated with Antimicrobial susceptibility of isolates from a range of a significantly higher MIC of amoxicillin (P¼0.011), but not other sources b-lactam antibiotics. Similarly, significant associations were ap- The MIC results are summarized in Table 1. Clindamycin (56%), parent between prescription of a cephalosporin [prescription tetracycline (49%) and co-amoxiclav (21%) resistance was (n¼11) versus no prescription (n¼58)] and higher MIC of ceftazi- highest in the UK CF Prevotella isolates, with reduced susceptibility dime (P¼0.009) and prescription of a tetracycline and higher to amoxicillin and ceftazidime also apparent in each group. All MICs of tetracycline (P¼0.007) and doxycycline (P¼0.043).

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Table 1. In vitro antimicrobial susceptibilities of clinical Prevotella isolates

Percentage of isolates with MIC (mg/L) indicated susceptibility

Prevotella group and antimicrobial agent range 50% 90% S I R

UK CF (number of isolates tested) amoxicillin (56) ,0.016 to .256 6 .256 NA NA NA azithromycin (47) 0.19 to .256 48 .256 NA NA NA ceftazidime (57) 0.094 to .256 6 .256 NA NA NA chloramphenicol (40) 0.19–4 1 4 100 0 0 clindamycin (57) ,0.016 to .256 .256 .256 44 0 56 co-amoxiclav (57) ,0.016 to .256 1 64 68 11 21 doxycycline (37) 0.047–24 1.5 6 NA NA NA meropenem (57) 0.004–0.38 0.064 0.125 100 0 0 metronidazole (57) ,0.016 to .256 0.125 0.38 96 0 4 piperacillin/tazobactam (57) ,0.016–4 0.023 1.5 100 0 0 tetracycline (41) ,0.016 to .256 12 128 36 15 49 tobramycin (57) 64 to .1024 .1024 .1024 NA NA NA

US CF (number of isolates tested) amoxicillin (20) 1 to .256 24 .256 NA NA NA azithromycin (19) 0.064 to .256 2 .256 NA NA NA ceftazidime (19) 0.5 to .256 8 96 NA NA NA chloramphenicol (18) 0.38–3 1 2 100 0 0 clindamycin (22) ,0.016 to .256 ,0.016 .256 59 0 41 co-amoxiclav (22) 0.064–2 0.5 1.5 100 0 0 doxycycline (21) 0.016 to .256 0.094 4 NA NA NA meropenem (20) 0.004–0.19 0.032 0.064 100 0 0 metronidazole (19) ,0.016–1.5 0.064 0.25 100 0 0 piperacillin/tazobactam (22) ,0.016–0.75 ,0.016 0.023 100 0 0 tetracycline (20) 0.125 to .256 0.38 24 60 20 20 tobramycin (22) .1024 .1024 .1024 NA NA NA

UK invasive (number of isolates tested) amoxicillin (48) ,0.016 to .256 16 .256 NA NA NA azithromycin (32) 0.125 to .256 0.75 .256 NA NA NA ceftazidime (48) 0.075 to .256 2 .256 NA NA NA chloramphenicol (31) 0.25–6 2 4 100 0 0 clindamycin (49) ,0.016 to .256 ,0.016 0.5 90 0 10 co-amoxiclav (48) ,0.016 to .256 0.25 6 88 4 8 doxycycline (34) 0.047–256 1.5 8 NA NA NA meropenem (48) 0.003–0.25 0.032 8 100 0 0 metronidazole (48) ,0.016 to .256 0.094 1 96 0 4 piperacillin/tazobactam (50) ,0.016–1.5 0.023 0.38 100 0 0 tetracycline (31) 0.19–256 1 64 51 10 39 tobramycin (49) 128 to .1024 .1024 .1024 NA NA NA

UK healthy control (number of isolates tested) amoxicillin (27) ,0.016 to .256 2 128 NA NA NA azithromycin (17) 0.064–4 1.5 3 NA NA NA ceftazidime (27) 0.125 to .256 0.75 32 NA NA NA chloramphenicol (17) 0.19–6 1.5 2 100 0 0 clindamycin (25) ,0.016 to .256 ,0.016 ,0.016 96 0 4 co-amoxiclav (25) ,0.016–3 0.047 3 100 0 0 doxycycline (16) 0.016–8 0.094 4 NA NA NA meropenem (26) 0.012–0.75 0.047 0.125 100 0 0 metronidazole (25) ,0.016–0.75 0.064 0.25 100 0 0

Continued

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Table 1. Continued

Percentage of isolates with MIC (mg/L) indicated susceptibility

Prevotella group and antimicrobial agent range 50% 90% S I R

piperacillin/tazobactam (25) ,0.016–1.5 0.023 0.75 100 0 0 tetracycline (16) 0.094–96 0.5 24 81 0 19 tobramycin (26) 128 to .1024 .1024 .1024 NA NA NA

S, susceptible; I, intermediate; R, resistant; NA, no anaerobic breakpoints approved by the CLSI. CLSI breakpoints (mg/L): chloramphenicol (S, ≤8; I, 16; R, ≥32), clindamycin (S, ≤2; I, 4; R, ≥8), co-amoxiclav (S, ≤4; I, 8; R, ≥16), meropenem (S, ≤4; I, 8; R, ≥16), metronidazole (S, ≤8; I, 16; R, ≥32), piperacillin/tazobactam (S, ≤32; I, 64; R, ≥128) and tetracycline (S, ≤4; I, 8; R, ≥16).

Table 2. Risk of antimicrobial non-susceptibility (resistance or P < 0.001 intermediate resistance) in UK CF Prevotella isolates relative to invasive P = 0.005 P = 0.011 Prevotella isolates 512.00 256.00 No. of non-susceptible 128.00 Median 64.00 isolates/no. of isolates 32.00 tested (%) 16.00 Relative risk 8.00 Antibiotic UK CF invasive (95% CI) P value 4.00 2.00 1.00 Chloramphenicol 0/40 (0) 0/31 (0) 0.50 Clindamycin 32/57 (56) 5/49 (10) 5.50 (2.32– ,0.001 0.25 13.02) 0.13 Co-amoxiclav 18/57 (32) 6/48 (12) 2.53 (1.09– 0.037 MIC (mg/L) Azithromycin 0.06 0.03 5.85) 0.02 Meropenem 0/57 (0) 0/48 (0) Metronidazole 2/57 (4) 2/48 (4) 0.84 (0.12– 1.000 5.76) Piperacillin/ 0/57 (0) 0/50 (0)

tazobactam Healthy controls Tetracycline 26/41 (64) 15/31 (49) 1.31 (0.85– 0.301 2.02) Current prescription (CF) No current prescription (CF)

Figure 1. Relationship between azithromycin prescription and MICs for Prevotella isolates from CF patients [current prescription (n¼30) versus b-Lactamase production and antimicrobial susceptibility no current prescription (n¼32)] and comparison with MICs for UK healthy ¼ Ninety-one of 153 isolates (59%) were b-lactamase positive, with control isolates (n 17). Any isolates recorded as having an MIC .256 mg/L are shown on the graph as an MIC of 512 mg/L. significantly higher concentrations of amoxicillin (P,0.001), ceftazidime (P,0.001), co-amoxiclav (P,0.001) and meropenem (P¼0.025) required to inhibit growth of b-lactamase-positive all isolates tested being susceptible. Previous studies have also versus b-lactamase-negative isolates. reported all Prevotella isolates as susceptible to piperacillin/ tazobactam,11,12 whilst meropenem resistance is rare11 and chloramphenicol resistance amongst Prevotella has yet to be 13 Discussion detected. Furthermore, metronidazole resistance was only detected in a small number of isolates. Co-amoxiclav resistance Prevotella spp. have been detected by culture in 30% of respira- (21%) was highest in the UK CF isolate group and exceeded the tory samples from CF patients,4 with molecular studies reporting levels reported by others.11,14,15 detection in a higher percentage of samples.7,8 Similarly, Prevotella When the three UK groups were compared, CF isolates demon- spp. are frequently detected as part of the normal airway micro- strated significantly higher MICs of both azithromycin and clinda- biota of healthy individuals.2,3 As Prevotella may potentially con- mycin than observed for invasive and healthy control isolates. tribute to infection and inflammation in the CF lung, we These resistance levels exceed those reported previously forclinical determined the susceptibility of clinical Prevotella isolates to a isolates from a range of body sites.11,14,15 Increased macrolide re- range of antibiotics. Meropenem, piperacillin/tazobactam and sistance amongst CF Prevotella isolates is similar to that previously chloramphenicol all demonstrated excellent in vitro activity, with reported for the CF-associated Streptococcus milleri group by

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Grinwis et al.,16 who linked increased macrolide resistance with Author contributions chronic exposure to azithromycin. We also found that current pre- L. J. S., M. M. T., M. S. M., D. F. G. and J. S. E. conceived and designed the re- scription of azithromycin was associated with significantly higher search, L. J. S., K. A. G., S. J. M., L. M. and J. H. performed the research, azithromycin MICs, corroborating the results of recent clinical L. J. S., M. M. T. and M. S. M. analysed the data, L. J. S., M. M. T., M. S. M., 9,17,18 trials in CF, non-CF bronchiectasis and COPD, which reported D. F. G. and J. S. E. wrote the paper, and M. C. W. and T. S. were responsible an association between chronic macrolide exposure and increased for review and revision of the paper. resistance amongst respiratory pathogens and members of the airway microbiota. As serum azithromycin levels could not be mea- sured in CF patients, we cannot conclude with absolute certainty the true exposure of Prevotella isolates to this antibiotic. Neverthe- Supplementary data less, our results indicate that azithromycin exposure may lead to increased resistance amongst organisms such as Prevotella resi- Tables S1 and S2 are available as Supplementary data at JAC Online (http:// jac.oxfordjournals.org/). dent in the CF airway microbiota. We also found that higher MICs of b-lactam/tetracycline antibiotics were associated with their prescription in the previous year, suggesting that resistance to these antibiotics amongst Prevotella in the CF airway microbiota is not intrinsic, but selected References by previous antibiotic treatment. Furthermore, 59% of isolates 1 Zaura E, Keijser BJ, Huse SM et al. Defining the healthy ‘core microbiome’ were positive for production of b-lactamases, which may shield of oral microbial communities. BMC Microbiol 2009; 9: 259. them and other bacteria from the effect of b-lactam antibiotics;19 2 Charlson ES, Bittinger K, Haas AR et al. Topographical continuity of higher concentrations of amoxicillin, ceftazidime, co-amoxiclav and bacterial populations in the healthy human respiratory tract. Am J Respir meropenem were required to inhibit growth of b-lactamase-positive Crit Care Med 2011; 184: 957–63. compared with b-lactamase-negative isolates. Therefore, to opti- 3 Tunney MM, Field TR, Moriarty TF et al. Detection of anaerobic bacteria in mize the treatment of CF pulmonary infection, whilst minimizing high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit the development of resistance, physicians may need to consider Care Med 2008; 177: 995–1001. the synergistic interactions that are occurring between bacteria 4 Tunney MM, Klem ER, Fodor AA et al. Use of culture and molecular within the polymicrobial lung microbiota. analysis to determine the effect of antibiotic treatment on microbial Inconclusion, this study has shownthat Prevotella isolatedfrom community diversity and abundance during exacerbation in patients with CF airways are significantly more resistant to azithromycin, clinda- cystic fibrosis. Thorax 2011; 66: 579–84. mycin and co-amoxiclav than non-CF isolates and that azithromy- 5 Harris JK, De GrooteMA, Sagel SD et al. Molecular identificationof bacteria cin resistance in CF isolates was associated with current prescription in bronchoalveolar lavage fluid from children with cystic fibrosis. Proc Natl of this macrolide. Further studies are required to determine when re- Acad Sci USA 2007; 104: 20529–33. sistancedevelops,ifitpersistsandifitimpactsonthe efficacyofanti- 6 Bittar F, Richet H, Dubus JCet al. Moleculardetection of multiple emerging biotic treatmentofCFpulmonaryinfection.Iftreatmentof Prevotella pathogens in sputa from cystic fibrosis patients. PLoS ONE 2008; 3: e2908. isindicatedinCF, ourfindings suggest that antibioticssuch as metro- 7 Zhao J, Schloss PD, Kalikin LM et al. Decade-long bacterial community nidazole, meropenem, piperacillin/tazobactam and chlorampheni- dynamics in cystic fibrosis airways. Proc Natl Acad Sci USA 2012; 109: col are likely to be most effective. 5809–14. 8 Fodor AA, Klem ER, Gilpin DF et al. The adult cystic fibrosis airway microbiota is stable over time and infection type, and highly resilient to antibiotic treatment of exacerbations. PLoS ONE 2012; 7: e45001. Acknowledgements 9 Elborn JS, Tunney MM. Macrolides and bronchiectasis: clinical benefit with We thank Mr Gerry McGrillen (School of Pharmacy, Queen’s University a resistance price. JAMA 2013; 309: 1295–6. Belfast, Belfast, UK) for technical assistance and Dr Valerie Hall (Anaerobe 10 Field TR, Sibley CD, Parkins MD et al. The genus Prevotella in cystic fibrosis Reference Laboratory, Cardiff, UK) for kindly providing invasive isolates. airways. Anaerobe 2010; 16: 337–44. 11 Jamal W, Shahin M, Rotimi VO. Surveillance and trends of antimicrobial resistance among clinical isolates of anaerobes in Kuwait hospitals from Funding 2002 to 2007. Anaerobe 2010; 16:1–5. This work was supported by grants from the HSC Research and Develop- 12 Citron DM, Goldstein EJ, Merriam CV et al. Bacteriology of ment, Public Health Agency, Northern Ireland, the Medical Research moderate-to-severe diabetic foot infections and in vitro activity of Council and the United States National Institutes of Health (HL092964, antimicrobial agents. J Clin Microbiol 2007; 45: 2819–28. HL084934 and 5R01 HL092964-04) through a US-Ireland Partnership 13 Liu CY, Huang YT, Liao CH et al. Increasing trends in antimicrobial Grant. M. M. T. was supported by a Health and Social Care Research and De- resistance among clinically important anaerobes and Bacteroides fragilis velopment, Public Health Agency, Northern Ireland-funded UK National In- isolates causing nosocomial infections: emerging resistance to stitute for Health Research Career Scientist Award. L. J. S. and K. A. G. were carbapenems. Antimicrob Agents Chemother 2008; 52: 3161–8. supported by Department of Employment and Learning, Northern Ireland, Studentships. 14 Ardila CM, Granada MI, Guzman IC. Antibiotic resistance of subgingival species in chronic periodontitis patients. J Periodontal Res 2010; 45: 557–63. 15 Lakhssassi N, Elhajoui N, Lodter JP et al. Antimicrobial susceptibility Transparency declarations variation of 50 anaerobic periopathogens in aggressive periodontitis: an None to declare. interindividual variability study. Oral Microbiol Immunol 2005; 20: 244–52.

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