AAC Accepts, published online ahead of print on 29 April 2013 Antimicrob. Agents Chemother. doi:10.1128/aac.00463-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Antimicrobial Susceptibility of Propionibacterium acnes Isolates from Shoulder Surgery John. K. Crane 1,3,#, Donald W. Hohman 2,3, Scott R. Nodzo 2,3, and Thomas R. Duquin 2,3 1 Department of Medicine, Division of Infectious Diseases, University at Buffalo, Buffalo, NY, USA 2 Department of Orthopaedic Surgery, University at Buffalo, Buffalo, NY and 3 Erie County Medical Center, Buffalo, NY. Running Title: P. acnes Susceptibilites # Address correspondence to: John K. Crane, MD, PhD Division of Infectious Diseases Room 317 Biomedical Research Bldg. 3435 Main St. Buffalo, NY 14214 716-829-2676 FAX 716-829-3889 jcrane@buffalo.edu Keywords: shoulder surgery orthopedic surgery hemolysis anaerobic 1
43 44 45 46 47 48 49 50 51 Abstract Orthopedic surgeons at our institution have noticed an increase in the number of infections due to Propionibacterium acnes, especially following operations on the shoulder. We collected P. acnes isolates from our hospital microbiology laboratory for one year and performed antimicrobial susceptibility testing on 28 strains from the shoulder. Antibiotics with the lowest MIC values against P. acnes included (MIC 50 and MIC 90) penicillin G (0.006, 0.125), cephalothin (0.047 and 0.094), and ceftriaxone (0.016, 0.045), while others also showed activity. Strains resistant to clindamycin were noted. 52 2
53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 Introduction. Propionibacterium acnes has been recognized as a significant and emerging pathogen in orthopedic surgery over the last ten years, especially after operations on the shoulder, and especially following shoulder arthroplasty with prosthetic material (1-3). At our institution, the number of recognized cases of P. acnes has increased noticeably over the last few years. Formulating treatment recommendations for patients with P. acnes orthopedic infections can be difficult, however, given the paucity of data on antibiotic susceptibility patterns. Many of the early reports on P. acnes antibiotic susceptibilities are from patients with facial acne attending dermatology clinics. Other studies are now decades old (4, 5) or originate from countries where antibiotic usage patterns are much different from those in North America (6, 7), leading us to question whether we could rely on those reports. Methods. We performed testing on 33 strains of P. acnes collected between Nov., 2010, and Dec., 2012, 28 of which were from orthopedic surgeries on the shoulder and 5 of which were isolates from bloodstream or other deep infections. P. acnes was identified using the Microscan Rapid Anaerobe ID method (Siemens Healthcare, W. Sacramento, CA). We tested this strain collection against a panel of ten antibiotics, with an emphasis on antibiotics that might actually be used to treat post-operative orthopedic infections. We used the E-test method (Biomerieux, Durham, NC) on Anaerobic Brucella blood agar. Two previous studies indicated that MICs obtained by E-test correlated well with results obtained using the agar dilution method (8, 9) and other experts consider the E-test an accepted method (10). E-test results obtained using Anaerobic Brucella blood agar were compared to those obtained using CDC anaerobe blood agar on a subset of 6 strains. 3
77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 Inocula were prepared to a 0.5 MacFarland standard from 48 h growth on anaerobic blood agar. Blood agars were from Anaerobe Systems (Morgan Hill, CA). We only tested a single isolate from each patient, although most patients had more than one positive cultures. P. acnes was cultured anaerobically at 37 º C for 48-72 hours using the GasPak EZ Anaerobe Container system (BD Corp., Franklin Lakes, NJ). Results. The results of our study are reported in Table 1. MICs measured on Brucella Blood agar were the same as those measured using CDC Anaerobe agar. The penicillins (penicillin G and amoxicillin) and cephalosporins (cephalothin and ceftriaxone) showed strong activity against P. acnes, with many isolates showing MICs lower than the lowest antibiotic concentration on the E-test strip. For example, for 15 of 28 isolates the MIC of amoxicillin was less than 0.016, the lowest concentration on the E-test strip. The strong activity of benzylpenicillin (penicillin G) against P. acnes was noted by Oprica et al. (11), but is not often considered as an option by orthopedic surgeons. We expected that moxifloxacin would show much greater activity against P. acnes than ciprofloxacin, but were surprised to see that moxifloxacin s MIC 50 and MIC 90 (0.125, 0.38) were only about 2- fold lower than ciprofloxacin (0.25, 0.5). Vancomycin is often viewed as a first-choice drug for post-operative infection in orthopedics due to its activity against Staphylococci, but its activity against P. acnes in our study was only fair (MICs 0.38, 0.5). This is especially relevant since the concentration of vancomycin required to eradicate P. acnes in an established biofilm (biofilm eradication concentration) was 128 µg/ml in recent studies (12, 13). Clindamycin was active against many P. acnes strains, but some strains were highly resistant (Table 1 and Fig. 1; clindamycin MICs 0.032, 8.5). A histogram of the distribution of MICs for clindamycin and linezolid showed distributions that appeared to be 4
100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 bimodal (Fig. 1), a pattern not seen with the other antibiotics tested (data not shown). Although our strain collection may be too small to definitely conclude there is a bimodal distribution, the presence of a subset of highly clindamycin-resistant P. acnes strains has also been noted in other studies of P. acnes, including collections of isolates from skin and deep systemic infection (11, 14). Ertapenem (MICs 0.032, 0.141) also showed good activity against P. acnes, which is notable because its once-daily administration makes home intravenous antibiotic administration more feasible. In addition, many patients who are reportedly allergic to penicillin are able to tolerate carbapenems. The MIC 50 values of the shoulder isolates were less than or equal to the MIC 50 from the non-shoulder isolates for all antibiotics tested. For example, the shoulder isolates showed an MIC 50 value to vancomycin of 0.38 mg/l compared to an MIC 50 of 1.5 for the non-shoulder isolates. (The non-shoulder isolates included one isolate each from bloodstream, tibia, hip prosthesis, cornea, and mediastinal lymph node.) Larger numbers of non-shoulder isolates, however, are needed to make definitive comparisons of antibiotic susceptibility between different body sites. Although MICs obtained with CDC Anaerobe agar and Brucella blood agar were identical, we noticed one difference between the two agar types. Some P. acnes strains were strongly hemolytic on Brucella blood agar, but these strains did not show hemolysis on CDC anaerobe agar, even though both agars contain 5% sheep blood. P. acnes strains showing hemolysis on Brucella blood agar appeared to have a more aggressive clinical course than the non-hemolytic strains. Our investigation on the role of hemolysis in P. acnes is being submitted for publication elsewhere. 5
123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 Guidelines for treatment of deep-seated P. acnes infections, including prosthetic joint infections, are few and are based mostly on anecdotal experience. In most reports, clindamycin and vancomycin are mentioned as the first drugs to consider for deep-seated P. acnes infections (15). Our data, like the study by Oprica et al. (11), suggests that penicillins and first-generation cephalosporins (cefazolin and cephalothin) show promise and should have an expanded role in studies of clinical efficacy. The variability in P. acnes susceptibility to clindamycin argues for routine antimicrobial susceptibility testing to clindamycin and other antibiotics. In addition, an expanded role for oral antibiotics should be considered in treatment of P. acnes infections, since many of the drugs that we found to be highly active in vitro are also highly bioavailable, including amoxicillin, moxifloxacin, and linezolid. Levy et al. advocated for a greater use of oral antibiotics, such as an oral ß- lactam plus rifampin, for treatment of P. acnes prosthetic infections (2) but actual practice has lagged behind these concepts, partly because of widely held biases in favor of intravenous treatment, and also due to the lack of controlled clinical studies. Further work in this area could result in improved outcomes of P. acnes infections at substantially lower costs than traditional approaches. 139 140 141 142 143 144 (Word count: 1030) Acknowledgements. This work was supported by a grant from Research for Health in Erie County. We thank Dr. Daniel Amsterdam, Department of Laboratory Medicine, and Ms. Lynn Connors, Bacteriology Supervisor, for their support and assistance in this project. We thank Dr. 6
145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 Philip Stegemann and Ms. Cathy Buyea, Department of Orthopaedics, for encouragement and for administrative and grant-writing support. Legend to Fig. 1. Fig. 1. Histogram of MIC Values for clindamycin and linezolid from the orthopedic isolates. MIC values for the strains are displayed as a histogram, with the number of each isolates falling into each bin of the MIC ranges shown. Panel A, clindamycin; panel B, linezolid. 7
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Table 1. MIC Values of P. acnes strains. (mg/l) Orthopedic Isolates from the Shoulder, n= 28 CLSI Breakpoint, mg/l MIC 50 MIC 90 Amoxicillin 0.028 0.117 -- a -- Penicillin G 0.006 0.125 0.5 4 Cephalothin 0.047 0.094 -- -- Ceftriaxone 0.016 0.045 -- -- Clindamycin 0.032 8.5 2 7 Ciprofloxacin 0.25 0.5 -- -- Moxifloxacin 0.125 0.38 2 0 Ertapenem 0.032 0.141 4 0 Vancomycin 0.38 0.5 -- -- Linezolid 0.25 0.93 -- -- % of strains resistant a, no interpretive standards from the Clinical Laboratory Standards Institute (CLSI).