Ceftolozane-Tazobactam Activity against Phylogenetically Diverse Clostridium difficile Strains Mark D. Gonzalez,a Meghan A. Wallace,a Tiffany Hink,b Erik R. Dubberke,b
Carey-Ann D. Burnhama
Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, Washington University School of Medicine, St. Louis, Missouri, USAa; Division of Infectious Diseases, Washington University School of Medicine, St. Louis, Missouri, USAb
Ceftolozane-tazobactam (C/T) is approved for the treatment of complicated intra-abdominal and urinary tract infections and has varied activity against anaerobic bacteria. Here, we evaluate the activity of C/T against a phylogenetically diverse collection of Clostridium difficile isolates and report uniformly high MICs (>256 g/ml) to C/T.
I
n December of 2014, ceftolozane-tazobactam received U.S. Food and Drug Administration (FDA) approval for the treatment of complicated intra-abdominal infections (cIAI) and complicated urinary tract infections (cUTI) (http://www.fda.gov /NewsEvents/Newsroom/PressAnnouncements/ucm427534.htm). Ceftolozane-tazobactam is a novel -lactam/-lactamase inhibitor combination with activity against Pseudomonas aeruginosa, extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae, and members of the Streptococcus anginosus group (1). The activity of ceftolozane-tazobactam against anaerobic bacteria is varied. For example, the MIC90 of 244 Bacteroides fragilis isolates, 86 Bacteroides thetaiotaomicron isolates, and 12 Fusobacterium species isolates was 4 g/ml, 32 g/ml, and 0.25 g/ml, respectively (2). Clostridium difficile infection (CDI) is well documented as a potential adverse consequence of antimicrobial therapy with a cephalosporin (3–5). The incidence and severity of CDI have increased in recent years, resulting in significant morbidity, mortality, and cost to the health care system (6, 7). C. difficile is classified as one of only three urgent, or highest priority, multidrug-resistant microbial threats by the Centers for Disease Control and Prevention (http://www.cdc.gov/drugresistance/threat-report-2013/). To date, limited clinical data regarding ceftolozane-tazobactam therapy exist, and the risk of CDI following therapy with this specific agent is relatively understudied. In two of the clinical trials that evaluated ceftolozane-tazobactam treatment of cIAI (8) and cUTI (9), 3 cases of CDI were documented (⬍1% incidence). In light of the varied antianaerobic activity of ceftolozane-tazobactam, an assessment of the antimicrobial resistance profile for ceftolozane-tazobactam with C. difficile could be important for understanding the potential risk of developing CDI during therapy with this agent. A recent study by Snydman et al. (2) evaluated the activity of ceftolozane-tazobactam against a large collection of anaerobic bacteria using agar dilution; this collection included 30 isolates of C. difficile. The MIC range of ceftolozane-tazobactam against the C. difficile isolates was 0.25 g/ml to ⱖ256 g/ml, and the MIC50 was ⱖ256 g/ml (2). In contrast, the MIC range and MIC50 documented for Clostridium perfringens (n ⫽ 11) and other Clostridium spp. (n ⫽ 13) were lower (MIC50, 0.25 g/ml and 16 g/ml, respectively) (2). Currently, there are no categorical interpretative criteria for ceftolozane-tazobactam MICs for Clostridium species. It is well documented that the overall antimicrobial resistance profiles of C. difficile strains can vary widely among different strain types (10–12). Unfortunately, no C. difficile strain
7084
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typing data were available as part of the Snydman et al. study (2), and thus it was unknown if this profile was generalizable to different C. difficile strain types. The objective of our study was to evaluate the antimicrobial activity of ceftolozane-tazobactam against a diverse collection of C. difficile strain types. The isolates investigated were recovered from fecal specimens or rectal swab specimens from patients at Barnes-Jewish Hospital from January 2010 to July 2012. Specimens were collected as part of other ongoing studies to assess C. difficile diagnostic assays, C. difficile infection, or asymptomatic colonization (3, 10, 13, 14). C. difficile isolates were recovered in culture (15) and ribotyped (16), as previously described. All isolates were recovered from frozen stocks and were subcultured twice on prereduced 5% sheep blood agar (Hardy Diagnostics, Santa Maria, CA) in an anaerobic environment prior to testing. The identity of each C. difficile isolate was confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using the Vitek MS (IVD version 2.3.3; bioMérieux, Durham, NC) (17). Ceftolozane-tazobactam susceptibility testing was performed using a gradient diffusion method (Etest; bioMérieux, Durham, NC), according to the manufacturer’s recommendations. In brief, a 1 McFarland standard suspension of each isolate was prepared in 0.9% saline and plated as a lawn onto brucella blood agar with hemin and vitamin K (Hardy Diagnostics), and ceftolozane-tazobactam Etest strips were applied. The plates were incubated under anaerobic conditions and read at 24 and 72 h of incubation. During each day of analysis, the quality control strain B. fragilis ATCC 25285 (acceptable quality control [QC] range, 0.12 to 1 g/ml) was tested. Eighty-one C. difficile isolates were tested, representing 15 different ribotypes (Table 1). Of note, our test set included 26 C. difficile ribotype 027 (NAP1/BI/ST-1) strains and 3 C. difficile ri-
Received 13 July 2015 Returned for modification 6 August 2015 Accepted 8 August 2015 Accepted manuscript posted online 17 August 2015 Citation Gonzalez MD, Wallace MA, Hink T, Dubberke ER, Burnham C-AD. 2015. Ceftolozane-tazobactam activity against phylogenetically diverse Clostridium difficile strains. Antimicrob Agents Chemother 59:7084 –7085. doi:10.1128/AAC.01670-15. Address correspondence to Carey-Ann D. Burnham, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Antimicrobial Agents and Chemotherapy
November 2015 Volume 59 Number 11
Ceftolozane-Tazobactam Activity against C. difficile
TABLE 1 Ribotypes and toxigenic statuses of evaluated C. difficile isolates (n ⫽ 81) C. difficile ribotype
No. of isolates tested
Toxin producing?
005 010 017 027 053 056 077 078 001/VPI/077/087 106/174 012-like 014/020 015/046 002/075 005/075
6 4 2 26 4 1 5 3 4 3 4 6 4 8 1
Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Total
81
4.
5.
6. 7.
8.
9.
botype 078 isolates. These strain types are currently among the most common ribotypes in North America and parts of Europe (18, 19); the toxigenic status of each ribotype was determined by phenotypic (i.e., toxin enzyme immunoassays) and/or genotypic methods (3, 10, 13, 14). Ceftolozane-tazobactam demonstrated no activity against any of the isolates investigated. All isolates tested (n ⫽ 81) had ceftolozane-tazobactam MICs of ⱖ256 g/ml at both the 24- and 72-h incubation time points. This phenotype appears to be distinct from that of other Clostridium species (2). A potential limitation of this study is that all of the isolates investigated were recovered at a single medical center. However, this limitation is mitigated by the careful characterization of the isolates, which demonstrated that a phylogenetically diverse population of strains was tested, and this is a strength of this investigation. The impact of ceftolozane-tazobactam on the gastrointestinal microbiota is unknown at this time. Similar to other cephalosporins that are documented to be risk factors for developing CDI (3–5), ceftolozane-tazobactam is predominantly eliminated by renal clearance (17). The concentration of this agent in the stool or the gastrointestinal tract is not known at this time. In conclusion, we have demonstrated that C. difficile appears to be uniformly resistant to ceftolozane-tazobactam. Additional clinical data are needed to appreciate the impact of this agent on both the intestinal microbiota and the relative risk of developing CDI during therapy.
10.
11. 12.
13. 14.
15.
16.
17.
ACKNOWLEDGMENT The Etest strips used in this investigation were provided by Cubist.
REFERENCES 1. Cho JC, Fiorenza MA, Estrada SJ. 2015. Ceftolozane-tazobactam: a novel cephalosporin/-lactamase inhibitor combination. Pharmacotherapy 35: 701–715. http://dx.doi.org/10.1002/phar.1609. 2. Snydman DR, McDermott LA, Jacobus NV. 2014. Activity of ceftolozane-tazobactam against a broad spectrum of recent clinical anaerobic isolates. Antimicrob Agents Chemother 58:1218 –1223. http://dx.doi.org /10.1128/AAC.02253-13. 3. Dubberke ER, Reske KA, Seiler S, Hink T, Kwon JH, Burnham CA.
November 2015 Volume 59 Number 11
18.
19.
2015. Risk factors for acquisition and loss of C. difficile colonization in hospitalized patients. Antimicrob Agents Chemother 59:4533– 4543. http: //dx.doi.org/10.1128/AAC.00642-15. Dubberke ER, Reske KA, Yan Y, Olsen MA, McDonald LC, Fraser VJ. 2007. Clostridium difficile-associated disease in a setting of endemicity: identification of novel risk factors. Clin Infect Dis 45:1543–1549. http://dx .doi.org/10.1086/523582. Slimings C, Riley TV. 2014. Antibiotics and hospital-acquired Clostridium difficile infection: update of systematic review and meta-analysis. J Antimicrob Chemother 69:881– 891. http://dx.doi.org/10.1093/jac /dkt477. Dubberke ER, Olsen MA. 2012. Burden of Clostridium difficile on the healthcare system. Clin Infect Dis 55(Suppl 2):S88 –S92. http://dx.doi.org /10.1093/cid/cis335. Hall AJ, Curns AT, McDonald LC, Parashar UD, Lopman BA. 2012. The roles of Clostridium difficile and norovirus among gastroenteritisassociated deaths in the United States, 1999 –2007. Clin Infect Dis 55:216 – 223. http://dx.doi.org/10.1093/cid/cis386. Solomkin J, Hershberger E, Miller B, Popejoy M, Friedland I, Steenbergen J, Yoon M, Collins S, Yuan G, Barie PS, Eckmann C. 2015. Ceftolozane-tazobactam plus metronidazole for complicated intraabdominal infections in an era of multidrug resistance: results from a randomized, double-blind, phase 3 trial (ASPECT-cIAI). Clin Infect Dis 60:1462–1471. http://dx.doi.org/10.1093/cid/civ097. Wagenlehner FM, Umeh O, Steenbergen J, Yuan G, Darouiche RO. 2015. Ceftolozane-tazobactam compared with levofloxacin in the treatment of complicated urinary-tract infections, including pyelonephritis: a randomised, double-blind, phase 3 trial (ASPECT-cUTI). Lancet 385: 1949 –1956. http://dx.doi.org/10.1016/S0140-6736(14)62220-0. Zhou Y, Burnham CA, Hink T, Chen L, Shaikh N, Wollam A, Sodergren E, Weinstock GM, Tarr PI, Dubberke ER. 2014. Phenotypic and genotypic analysis of Clostridium difficile isolates: a single-center study. J Clin Microbiol 52:4260 – 4266. http://dx.doi.org/10.1128/JCM.02115-14. Tenover FC, Tickler IA, Persing DH. 2012. Antimicrobial-resistant strains of Clostridium difficile from North America. Antimicrob Agents Chemother 56:2929 –2932. http://dx.doi.org/10.1128/AAC.00220-12. Spigaglia P, Barbanti F, Mastrantonio P, European Study Group of Clostridium difficile (ESGCD). 2011. Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother 66:2227– 2234. http://dx.doi.org/10.1093/jac/dkr292. Alasmari F, Seiler SM, Hink T, Burnham CA, Dubberke ER. 2014. Prevalence and risk factors for asymptomatic Clostridium difficile carriage. Clin Infect Dis 59:216 –222. http://dx.doi.org/10.1093/cid/ciu258. Dubberke ER, Han Z, Bobo L, Hink T, Lawrence B, Copper S, Hoppe-Bauer J, Burnham C-AD, Dunne WM, Jr. 2011. Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections. J Clin Microbiol 49:2887–2893. http://dx.doi.org /10.1128/JCM.00891-11. Hink T, Burnham CA, Dubberke ER. 2013. A systematic evaluation of methods to optimize culture-based recovery of Clostridium difficile from stool specimens. Anaerobe 19:39 – 43. http://dx.doi.org/10.1016/j .anaerobe.2012.12.001. Westblade LF, Chamberland RR, MacCannell D, Collins R, Dubberke ER, Dunne WM, Jr, Burnham CA. 2013. Development and evaluation of a novel, semiautomated Clostridium difficile typing platform. J Clin Microbiol 51:621– 624. http://dx.doi.org/10.1128/JCM.02627-12. Garner O, Mochon A, Branda J, Burnham CA, Bythrow M, Ferraro M, Ginocchio C, Jennemann R, Manji R, Procop GW, Richter S, Rychert J, Sercia L, Westblade L, Lewinski M. 2014. Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK MS system. Clin Microbiol Infect 20:335–339. http://dx.doi.org/10.1111 /1469-0691.12317. Freeman J, Bauer MP, Baines SD, Corver J, Fawley WN, Goorhuis B, Kuijper EJ, Wilcox MH. 2010. The changing epidemiology of Clostridium difficile infections. Clin Microbiol Rev 23:529 –549. http://dx.doi.org/10 .1128/CMR.00082-09. Kuijper EJ, Coignard B, Tüll P, ESCMID Study Group for Clostridium difficile, EU Member States, European Centre for Disease Prevention and Control. 2006. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 12(Suppl 6):S2–S18.
Antimicrobial Agents and Chemotherapy
aac.asm.org
7085
Carey-Ann D. Burnhama
Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, Washington University School of Medicine, St. Louis, Missouri, USAa; Division of Infectious Diseases, Washington University School of Medicine, St. Louis, Missouri, USAb
Ceftolozane-tazobactam (C/T) is approved for the treatment of complicated intra-abdominal and urinary tract infections and has varied activity against anaerobic bacteria. Here, we evaluate the activity of C/T against a phylogenetically diverse collection of Clostridium difficile isolates and report uniformly high MICs (>256 g/ml) to C/T.
I
n December of 2014, ceftolozane-tazobactam received U.S. Food and Drug Administration (FDA) approval for the treatment of complicated intra-abdominal infections (cIAI) and complicated urinary tract infections (cUTI) (http://www.fda.gov /NewsEvents/Newsroom/PressAnnouncements/ucm427534.htm). Ceftolozane-tazobactam is a novel -lactam/-lactamase inhibitor combination with activity against Pseudomonas aeruginosa, extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae, and members of the Streptococcus anginosus group (1). The activity of ceftolozane-tazobactam against anaerobic bacteria is varied. For example, the MIC90 of 244 Bacteroides fragilis isolates, 86 Bacteroides thetaiotaomicron isolates, and 12 Fusobacterium species isolates was 4 g/ml, 32 g/ml, and 0.25 g/ml, respectively (2). Clostridium difficile infection (CDI) is well documented as a potential adverse consequence of antimicrobial therapy with a cephalosporin (3–5). The incidence and severity of CDI have increased in recent years, resulting in significant morbidity, mortality, and cost to the health care system (6, 7). C. difficile is classified as one of only three urgent, or highest priority, multidrug-resistant microbial threats by the Centers for Disease Control and Prevention (http://www.cdc.gov/drugresistance/threat-report-2013/). To date, limited clinical data regarding ceftolozane-tazobactam therapy exist, and the risk of CDI following therapy with this specific agent is relatively understudied. In two of the clinical trials that evaluated ceftolozane-tazobactam treatment of cIAI (8) and cUTI (9), 3 cases of CDI were documented (⬍1% incidence). In light of the varied antianaerobic activity of ceftolozane-tazobactam, an assessment of the antimicrobial resistance profile for ceftolozane-tazobactam with C. difficile could be important for understanding the potential risk of developing CDI during therapy with this agent. A recent study by Snydman et al. (2) evaluated the activity of ceftolozane-tazobactam against a large collection of anaerobic bacteria using agar dilution; this collection included 30 isolates of C. difficile. The MIC range of ceftolozane-tazobactam against the C. difficile isolates was 0.25 g/ml to ⱖ256 g/ml, and the MIC50 was ⱖ256 g/ml (2). In contrast, the MIC range and MIC50 documented for Clostridium perfringens (n ⫽ 11) and other Clostridium spp. (n ⫽ 13) were lower (MIC50, 0.25 g/ml and 16 g/ml, respectively) (2). Currently, there are no categorical interpretative criteria for ceftolozane-tazobactam MICs for Clostridium species. It is well documented that the overall antimicrobial resistance profiles of C. difficile strains can vary widely among different strain types (10–12). Unfortunately, no C. difficile strain
7084
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typing data were available as part of the Snydman et al. study (2), and thus it was unknown if this profile was generalizable to different C. difficile strain types. The objective of our study was to evaluate the antimicrobial activity of ceftolozane-tazobactam against a diverse collection of C. difficile strain types. The isolates investigated were recovered from fecal specimens or rectal swab specimens from patients at Barnes-Jewish Hospital from January 2010 to July 2012. Specimens were collected as part of other ongoing studies to assess C. difficile diagnostic assays, C. difficile infection, or asymptomatic colonization (3, 10, 13, 14). C. difficile isolates were recovered in culture (15) and ribotyped (16), as previously described. All isolates were recovered from frozen stocks and were subcultured twice on prereduced 5% sheep blood agar (Hardy Diagnostics, Santa Maria, CA) in an anaerobic environment prior to testing. The identity of each C. difficile isolate was confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using the Vitek MS (IVD version 2.3.3; bioMérieux, Durham, NC) (17). Ceftolozane-tazobactam susceptibility testing was performed using a gradient diffusion method (Etest; bioMérieux, Durham, NC), according to the manufacturer’s recommendations. In brief, a 1 McFarland standard suspension of each isolate was prepared in 0.9% saline and plated as a lawn onto brucella blood agar with hemin and vitamin K (Hardy Diagnostics), and ceftolozane-tazobactam Etest strips were applied. The plates were incubated under anaerobic conditions and read at 24 and 72 h of incubation. During each day of analysis, the quality control strain B. fragilis ATCC 25285 (acceptable quality control [QC] range, 0.12 to 1 g/ml) was tested. Eighty-one C. difficile isolates were tested, representing 15 different ribotypes (Table 1). Of note, our test set included 26 C. difficile ribotype 027 (NAP1/BI/ST-1) strains and 3 C. difficile ri-
Received 13 July 2015 Returned for modification 6 August 2015 Accepted 8 August 2015 Accepted manuscript posted online 17 August 2015 Citation Gonzalez MD, Wallace MA, Hink T, Dubberke ER, Burnham C-AD. 2015. Ceftolozane-tazobactam activity against phylogenetically diverse Clostridium difficile strains. Antimicrob Agents Chemother 59:7084 –7085. doi:10.1128/AAC.01670-15. Address correspondence to Carey-Ann D. Burnham, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Antimicrobial Agents and Chemotherapy
November 2015 Volume 59 Number 11
Ceftolozane-Tazobactam Activity against C. difficile
TABLE 1 Ribotypes and toxigenic statuses of evaluated C. difficile isolates (n ⫽ 81) C. difficile ribotype
No. of isolates tested
Toxin producing?
005 010 017 027 053 056 077 078 001/VPI/077/087 106/174 012-like 014/020 015/046 002/075 005/075
6 4 2 26 4 1 5 3 4 3 4 6 4 8 1
Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Total
81
4.
5.
6. 7.
8.
9.
botype 078 isolates. These strain types are currently among the most common ribotypes in North America and parts of Europe (18, 19); the toxigenic status of each ribotype was determined by phenotypic (i.e., toxin enzyme immunoassays) and/or genotypic methods (3, 10, 13, 14). Ceftolozane-tazobactam demonstrated no activity against any of the isolates investigated. All isolates tested (n ⫽ 81) had ceftolozane-tazobactam MICs of ⱖ256 g/ml at both the 24- and 72-h incubation time points. This phenotype appears to be distinct from that of other Clostridium species (2). A potential limitation of this study is that all of the isolates investigated were recovered at a single medical center. However, this limitation is mitigated by the careful characterization of the isolates, which demonstrated that a phylogenetically diverse population of strains was tested, and this is a strength of this investigation. The impact of ceftolozane-tazobactam on the gastrointestinal microbiota is unknown at this time. Similar to other cephalosporins that are documented to be risk factors for developing CDI (3–5), ceftolozane-tazobactam is predominantly eliminated by renal clearance (17). The concentration of this agent in the stool or the gastrointestinal tract is not known at this time. In conclusion, we have demonstrated that C. difficile appears to be uniformly resistant to ceftolozane-tazobactam. Additional clinical data are needed to appreciate the impact of this agent on both the intestinal microbiota and the relative risk of developing CDI during therapy.
10.
11. 12.
13. 14.
15.
16.
17.
ACKNOWLEDGMENT The Etest strips used in this investigation were provided by Cubist.
REFERENCES 1. Cho JC, Fiorenza MA, Estrada SJ. 2015. Ceftolozane-tazobactam: a novel cephalosporin/-lactamase inhibitor combination. Pharmacotherapy 35: 701–715. http://dx.doi.org/10.1002/phar.1609. 2. Snydman DR, McDermott LA, Jacobus NV. 2014. Activity of ceftolozane-tazobactam against a broad spectrum of recent clinical anaerobic isolates. Antimicrob Agents Chemother 58:1218 –1223. http://dx.doi.org /10.1128/AAC.02253-13. 3. Dubberke ER, Reske KA, Seiler S, Hink T, Kwon JH, Burnham CA.
November 2015 Volume 59 Number 11
18.
19.
2015. Risk factors for acquisition and loss of C. difficile colonization in hospitalized patients. Antimicrob Agents Chemother 59:4533– 4543. http: //dx.doi.org/10.1128/AAC.00642-15. Dubberke ER, Reske KA, Yan Y, Olsen MA, McDonald LC, Fraser VJ. 2007. Clostridium difficile-associated disease in a setting of endemicity: identification of novel risk factors. Clin Infect Dis 45:1543–1549. http://dx .doi.org/10.1086/523582. Slimings C, Riley TV. 2014. Antibiotics and hospital-acquired Clostridium difficile infection: update of systematic review and meta-analysis. J Antimicrob Chemother 69:881– 891. http://dx.doi.org/10.1093/jac /dkt477. Dubberke ER, Olsen MA. 2012. Burden of Clostridium difficile on the healthcare system. Clin Infect Dis 55(Suppl 2):S88 –S92. http://dx.doi.org /10.1093/cid/cis335. Hall AJ, Curns AT, McDonald LC, Parashar UD, Lopman BA. 2012. The roles of Clostridium difficile and norovirus among gastroenteritisassociated deaths in the United States, 1999 –2007. Clin Infect Dis 55:216 – 223. http://dx.doi.org/10.1093/cid/cis386. Solomkin J, Hershberger E, Miller B, Popejoy M, Friedland I, Steenbergen J, Yoon M, Collins S, Yuan G, Barie PS, Eckmann C. 2015. Ceftolozane-tazobactam plus metronidazole for complicated intraabdominal infections in an era of multidrug resistance: results from a randomized, double-blind, phase 3 trial (ASPECT-cIAI). Clin Infect Dis 60:1462–1471. http://dx.doi.org/10.1093/cid/civ097. Wagenlehner FM, Umeh O, Steenbergen J, Yuan G, Darouiche RO. 2015. Ceftolozane-tazobactam compared with levofloxacin in the treatment of complicated urinary-tract infections, including pyelonephritis: a randomised, double-blind, phase 3 trial (ASPECT-cUTI). Lancet 385: 1949 –1956. http://dx.doi.org/10.1016/S0140-6736(14)62220-0. Zhou Y, Burnham CA, Hink T, Chen L, Shaikh N, Wollam A, Sodergren E, Weinstock GM, Tarr PI, Dubberke ER. 2014. Phenotypic and genotypic analysis of Clostridium difficile isolates: a single-center study. J Clin Microbiol 52:4260 – 4266. http://dx.doi.org/10.1128/JCM.02115-14. Tenover FC, Tickler IA, Persing DH. 2012. Antimicrobial-resistant strains of Clostridium difficile from North America. Antimicrob Agents Chemother 56:2929 –2932. http://dx.doi.org/10.1128/AAC.00220-12. Spigaglia P, Barbanti F, Mastrantonio P, European Study Group of Clostridium difficile (ESGCD). 2011. Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother 66:2227– 2234. http://dx.doi.org/10.1093/jac/dkr292. Alasmari F, Seiler SM, Hink T, Burnham CA, Dubberke ER. 2014. Prevalence and risk factors for asymptomatic Clostridium difficile carriage. Clin Infect Dis 59:216 –222. http://dx.doi.org/10.1093/cid/ciu258. Dubberke ER, Han Z, Bobo L, Hink T, Lawrence B, Copper S, Hoppe-Bauer J, Burnham C-AD, Dunne WM, Jr. 2011. Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections. J Clin Microbiol 49:2887–2893. http://dx.doi.org /10.1128/JCM.00891-11. Hink T, Burnham CA, Dubberke ER. 2013. A systematic evaluation of methods to optimize culture-based recovery of Clostridium difficile from stool specimens. Anaerobe 19:39 – 43. http://dx.doi.org/10.1016/j .anaerobe.2012.12.001. Westblade LF, Chamberland RR, MacCannell D, Collins R, Dubberke ER, Dunne WM, Jr, Burnham CA. 2013. Development and evaluation of a novel, semiautomated Clostridium difficile typing platform. J Clin Microbiol 51:621– 624. http://dx.doi.org/10.1128/JCM.02627-12. Garner O, Mochon A, Branda J, Burnham CA, Bythrow M, Ferraro M, Ginocchio C, Jennemann R, Manji R, Procop GW, Richter S, Rychert J, Sercia L, Westblade L, Lewinski M. 2014. Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK MS system. Clin Microbiol Infect 20:335–339. http://dx.doi.org/10.1111 /1469-0691.12317. Freeman J, Bauer MP, Baines SD, Corver J, Fawley WN, Goorhuis B, Kuijper EJ, Wilcox MH. 2010. The changing epidemiology of Clostridium difficile infections. Clin Microbiol Rev 23:529 –549. http://dx.doi.org/10 .1128/CMR.00082-09. Kuijper EJ, Coignard B, Tüll P, ESCMID Study Group for Clostridium difficile, EU Member States, European Centre for Disease Prevention and Control. 2006. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 12(Suppl 6):S2–S18.
Antimicrobial Agents and Chemotherapy
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7085
