Accepted Manuscript Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia Kate E. Mackin, Briony Elliott, Despina Kotsanas, Benjamin P. Howden, Glen P. Carter, Tony M. Korman, Thomas V. Riley, Julian I. Rood, Grant A. Jenkin, Dena Lyras PII:
S1075-9964(15)30017-2
DOI:
10.1016/j.anaerobe.2015.05.001
Reference:
YANAE 1441
To appear in:
Anaerobe
Received Date: 24 January 2015 Revised Date:
24 April 2015
Accepted Date: 1 May 2015
Please cite this article as: Mackin KE, Elliott B, Kotsanas D, Howden BP, Carter GP, Korman TM, Riley TV, Rood JI, Jenkin GA, Lyras D, Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia, Anaerobe (2015), doi: 10.1016/ j.anaerobe.2015.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia
Carter a, Tony M. Korman
c,f
, Thomas V. Riley
b,g
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Kate E. Mackin a, Briony Elliott b, Despina Kotsanas c, Benjamin P. Howden d,e, Glen P. , Julian I. Rood a, Grant A. Jenkin c,
a
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and Dena Lyras a*
Department of Microbiology, Monash University, Clayton, VIC, Australia,
b
School of
Australia, d
c
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Pathology and Laboratory Medicine, The University of Western Australia, Nedlands, WA, Monash Infectious Diseases, Monash Health, Clayton, VIC, Australia,
Department of Microbiology, Austin Health, Heidelberg, VIC, Australia, e Department of
Microbiology and Immunology, University of Melbourne, VIC, Australia, f Department of
Nedlands, WA, Australia.
g
PathWest Laboratory Medicine,
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Microbiology, Monash Health, Clayton, VIC, Australia,
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*corresponding author: [email protected] phone: +61 3 9902 9155
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postal address: Department of Microbiology, Building 76, Monash University, Victoria, Australia 3800
1
ACCEPTED MANUSCRIPT ABSTRACT Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were
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susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only
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detected in recent strains.
HIGHLIGHTS:
C. difficile is a nosocomial pathogen, with different strain types causing disease.
•
We have generated a snap-shot of C. difficile strain types circulating in Victoria.
•
The diversity of isolates in Victoria may differ from other parts of the world.
•
Most strains were toxinotype 0, but toxinotypes V and VIII strains were also found.
•
Strains were metronidazole and vancomycin sensitive; 2 were moxifloxacin resistant.
BODY OF PAPER
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Clostridium difficile is the most commonly recognised cause of antibiotic-associated diarrhea in humans (1). The development of C. difficile infection (CDI) is linked to disruption of the
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host microbiota (2), which often occurs after antibiotic therapy. The major factors contributing to disease are toxins A and B (3). The genes encoding these toxins are found on a chromosomally-located region, the Pathogenicity Locus (PaLoc). Variations in the PaLoc may result in differences in the toxins produced or in their regulation. This variation is the basis of toxinotyping, which groups strains by different PCR-RFLP patterns in sections of the PaLoc (4). Some strains also produce a third toxin, CDT, which may act as a colonisation factor (5). Increased morbidity and mortality due to C. difficile worldwide over the last 2
ACCEPTED MANUSCRIPT decade has been linked to fluoroquinolone-resistant toxinotype III/ribotype 027 strains, which may be more virulent than other strain types (6, 7). The first case of locally-acquired ribotype 027 in Australia was identified in Melbourne, Victoria, in 2010 (8). In addition, suggestions that toxinotype V/ribotype 078 isolates may demonstrate increased virulence capacity (9, 10)
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are of concern, while toxinotype VIII/ribotype 017 isolates have also been associated with outbreaks of severe disease (11). The ability of strains to spread and persist in the healthcare setting means there is a need to monitor isolates for the emergence of these strain types in
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new locations. Antibiotic stewardship often plays a central role in infection control practices targeting C. difficile. Thus, knowledge of the strain types present in a given hospital and their
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antibiotic susceptibility profiles may be useful in targeting this organism. However, the only published study examining antibiotic resistance in Australia is from the state of New South Wales in 2002 (12), with no published research papers available regarding isolates from other
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parts of the country.
This study aimed to generate an understanding of the C. difficile strain types present in the state of Victoria at different times. Two healthcare networks contributed C. difficile isolates
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from patients (n = 123) during 2006-2009. In addition, 50 historic (1979-1989) C. difficile
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strains from Victoria were characterised. All strains were toxinotyped, to examine variation in the PaLoc (4), and non-toxigenic isolates were confirmed by PCR (13). PCR was used to screen isolates for the presence of cdtB (14), which encodes part of CDT, and the tcdC gene, encoding a toxin regulator, was sequenced (15) (Table 1). We found that 75.6% of recent and 68% of historic Victorian isolates were toxinotype 0, in line with previous studies in other countries where toxinotype 0 can comprise 75-80% of isolates (12, 16). Recent strains included toxinotypes V (5/123) and VIII (2/123); these types were not found in the older
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ACCEPTED MANUSCRIPT isolates of this study. No toxinotype III/ribotype 027 strains were identified in either time period.
Ribotyping analysis (17) revealed further diversity of strain types in Victoria, with
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approximately 31% of recent isolates belonging to ribotype 014; this type is often found elsewhere in the world (18, 19). The next most common ribotypes identified (6% each of recent isolates) were 054, which is not often found in other countries (18, 20), and a unique
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Australian ribotype with no major worldwide equivalent. This data suggests that the diversity of C. difficile clinical isolates in Victoria may differ from other parts of the world. Of the
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toxinotype V isolates, one each belonged to the closely-related ribotypes 126 and 127. Two other toxinotype V isolates were ribotype 078, confirming the presence of this strain type in Victoria.
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We also examined a selection of isolates for antimicrobial susceptibilities (n = 23). Nine of these isolates were historic strains while 14 were recent isolates (Tables 2 and 3). The antibiotics tested were bacitracin, clindamycin, chloramphenicol, fusidic acid, erythromycin,
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moxifloxacin, penicillin, tetracycline, teicoplanin, vancomycin and metronidazole. MICs
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were determined following Clinical and Laboratory Standards Institute (CLSI) guidelines for agar dilution, with resistance breakpoints based on CLSI recommendations for anaerobes (21) where available. The resistance breakpoint determined by The European Committee on Antimicrobial Susceptibility Testing (EUCAST) for vancomycin (>2 µg/ml) (clinical break points – bacteria v.3.0; http://www.eucast.org/clinical_breakpoints/) was used as there is no CLSI recommendation. Where possible, resistance mechanisms were identified by PCR and/or sequencing, using primers specific for erm(B) (22), tet(M) (23), tet(W) (24), tetA(P)
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ACCEPTED MANUSCRIPT (23), tetB(P) (23), tndX (25), int (5’ CAGGTCTTCGTATTTCAGAG 3’ and 5’ TGTATGTCGCAAACTATG 3’), gyrA (26) and gyrB (26).
In Australia, metronidazole is the recommended first-line treatment for non-severe CDI and
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vancomycin is recommended for severe cases and recurrent disease (27). All of the isolates tested in this study were susceptible to metronidazole and vancomycin. Alternatives suggested for the treatment of CDI include teicoplanin (28) fusidic acid (29), and bacitracin
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(30). All isolates tested in this study demonstrated low teicoplanin MICs. However, all showed high bacitracin MICs, and 35% of recent isolates had high MICs to fusidic acid.
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Resistance to both of these agents has been documented previously (31, 32), suggesting that these two antimicrobials may not be viable alternatives.
Clindamycin use is strongly associated with the development of CDI (33). All isolates tested,
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except one, were resistant to clindamycin. Five of these isolates also showed high erythromycin MICs. In C. difficile this resistance is often encoded by an erm(B) gene, which may be carried on a transposon (22, 34); three of these five isolates were positive for erm(B)
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by PCR. The other two isolates were negative in this PCR suggesting that another mechanism
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may be responsible for the reduced susceptibility of these strains. These isolates both demonstrated high erythromycin MICs (>32 µg/ml), but low-level clindamycin resistance (8 µg/ml).
One of the erm(B)-containing isolates demonstrated high MICs to many of the antimicrobial agents, showing resistance to fusidic acid, penicillin, and tetracycline. The tetracycline resistance determinant appears to be carried on Tn5397 with positive PCR results for both tet(M) and tndX, as the latter is specific to this element. Another isolate, a recent toxinotype 5
ACCEPTED MANUSCRIPT V/ribotype 078 strain, contains a Tn916-like element as indicated by positive PCR results for tet(M) and the Tn916-specific int gene; such elements have been found in this strain type previously (35). Tetracycline resistance in the Clostridia may be mediated by other tet genes
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(24, 36), however, these could not be detected in the third tetracycline-resistant isolate.
While the development of fluoroquinolone resistance has been associated with the spread of C. difficile ribotype 027 (5), it is seen in other strain types (37). In this study, decreased
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susceptibility to moxifloxacin (MIC = 16 µg/ml) was found in two recent isolates, while all of the historic isolates tested were fully susceptible. In C. difficile resistance to
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fluoroquinolones is mediated by mutations in the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB (26), with the most common change resulting in the substitution of threonine to isoleucine at position 82 of GyrA (37). This same substitution was found in
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the two isolates in this study.
The rates of C. difficile infection are increasing in Australia (38) and we have very limited information about the types of strains circulating in our population. While much of the focus
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has been on C. difficile ribotype 027, other strain types can cause outbreaks of disease, as
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recently seen in our local area (39). This study has identified the predominant C. difficile toxinotypes in two major Victorian healthcare networks during non-outbreak conditions, and demonstrated that these isolates may be resistant to many antimicrobial agents. Many of the isolates identified in this study were toxinotype 0, consistent with findings in other parts of the world. However, the results of this study are also consistent with the hypothesis that the epidemiology of C. difficile is different in Australia compared to other countries. Of particular concern is the identification of toxinotype V and toxinotype VIII strains in this study. 6
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The impact of variant C. difficile strain types abroad highlights why the prevention and control of this organism in Australian healthcare settings is of paramount importance. The recent move away from culturing for C. difficile towards other methods such as PCR or
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enzyme immunoassay means that important strain types cannot be detected if or when they do emerge in Australia, and antibiotic resistance properties cannot be determined. This is a concern not just in terms of imported strains posing a threat to Australian biosecurity, but also
ACKNOWLEDGMENTS
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in that uniquely Australian strain types could disseminate elsewhere.
The researchers were supported by Project Grant GNT1051584 from the Australian National
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Health and Medical Research Council (NHMRC) and Discovery Grant DP1093891 from the Australian Research Council (ARC). DL was supported by an ARC Future Fellowship (FT120100779) from the ARC. BH was supported by an NHMRC Career Development
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Fellowship (GNT1023526).
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ACCEPTED MANUSCRIPT REFERENCES
1.
McDonald LC, Owings M, Jernigan DB. 2006. Clostridium difficile infection in patients discharged from US short-stay hospitals, 1996-2003. Emerg Infect Dis
2.
RI PT
12:409-415.
Reeves AE, Theriot CM, Bergin IL, Huffnagle GB, Schloss PD, Young VB. 2011. The interplay between microbiome dynamics and pathogen dynamics in a murine
3.
SC
model of Clostridium difficile infection. Gut Microbes 2:145-158.
Carter GP, Awad MM, Kelly ML, Rood JI, Lyras D. 2011. TcdB or not TcdB: a
4.
M AN U
tale of two Clostridium difficile toxins. Future Microbiol 6:121-123. Rupnik M, Avesani V, Janc M, von Eichel-Streiber C, Delmee M. 1998. A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 36:2240-2247.
Schwan C, Stecher B, Tzivelekidis T, van Ham M, Rohde M, Hardt WD,
TE D
5.
Wehland J, Aktories K. 2009. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog
Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S, Bourgault
AC C
6.
EP
5:e1000626.
AM, Nguyen T, Frenette C, Kelly M, Vibien A, Brassard P, Fenn S, Dewar K, Hudson TJ, Horn R, Rene P, Monczak Y, Dascal A. 2005. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med 353:2442-2449.
7.
Rupnik M, Wilcox MH, Gerding DN. 2009. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7:526-536.
8
ACCEPTED MANUSCRIPT 8.
Richards M, Knox J, Elliott B, Mackin K, Lyras D, Waring LJ, Riley TV. 2011. Severe infection with Clostridium difficile PCR ribotype 027 acquired in Melbourne, Australia. Med J Aust 194:369-371.
9.
Goorhuis A, Bakker D, Corver J, Debast SB, Harmanus C, Notermans DW,
RI PT
Bergwerff AA, Dekker FW, Kuijper EJ. 2008. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis 47:1162-1170.
Knetsch CW, Hensgens MP, Harmanus C, van der Bijl MW, Savelkoul PH,
SC
10.
Kuijper EJ, Corver J, van Leeuwen HC. 2011. Genetic markers for Clostridium
11.
M AN U
difficile lineages linked to hypervirulence. Microbiology 157:3113-3123. Drudy D, Harnedy N, Fanning S, Hannan M, Kyne L. 2007. Emergence and control of fluoroquinolone-resistant, toxin A-negative, toxin B-positive Clostridium difficile. Infect Control Hosp Epidemiol 28:932-940.
Leroi MJ, Siarakas S, Gottlieb T. 2002. E test susceptibility testing of nosocomial
TE D
12.
Clostridium difficile isolates against metronidazole, vancomycin, fusidic acid and the
21:72-74.
Braun V, Hundsberger T, Leukel P, Sauerborn M, von Eichel-Streiber C. 1996.
AC C
13.
EP
novel agents moxifloxacin, gatifloxacin, and linezolid. Eur J Clin Microbiol Infect Dis
Definition of the single integration site of the pathogenicity locus in Clostridium
difficile. Gene 181:29-38.
14.
Stubbs S, Rupnik M, Gibert M, Brazier J, Duerden B, Popoff M. 2000.
Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile. FEMS Microbiol Lett 186:307-312.
9
ACCEPTED MANUSCRIPT 15.
Spigaglia P, Mastrantonio P. 2002. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol 40:3470-3475.
16.
Barbut F, Mastrantonio P, Delmee M, Brazier J, Kuijper E, Poxton I, European
RI PT
Study Group on Clostridium difficile. 2007. Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect 13:1048-1057.
Stubbs SL, Brazier JS, O'Neill GL, Duerden BI. 1999. PCR targeted to the 16S-
SC
17.
23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a
18.
M AN U
library consisting of 116 different PCR ribotypes. J Clin Microbiol 37:461-463. Bauer MP, Notermans DW, van Benthem BH, Brazier JS, Wilcox MH, Rupnik M, Monnet DL, van Dissel JT, Kuijper EJ, ECDIS Study Group. 2011. Clostridium difficile infection in Europe: a hospital-based survey. Lancet 377:63-73. Walk ST, Micic D, Jain R, Lo ES, Trivedi I, Liu EW, Almassalha LM, Ewing
TE D
19.
SA, Ring C, Galecki AT, Rogers MA, Washer L, Newton DW, Malani PN, Young VB, Aronoff DM. 2012. Clostridium difficile ribotype does not predict severe
Wilcox MH, Shetty N, Fawley WN, Shemko M, Coen P, Birtles A, Cairns M,
AC C
20.
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infection. Clin Infect Dis 55:1661-1668.
Curran MD, Dodgson KJ, Green SM, Hardy KJ, Hawkey PM, Magee JG, Sails AD, Wren MW. 2012. Changing epidemiology of Clostridium difficile infection
following the introduction of a national ribotyping-based surveillance scheme in England. Clin Infect Dis 55:1056-1063.
21.
Clinical and Laboratory Standards Institute. 2007. Methods for antimicrobial susceptibility testing of anaerobic bacteria; approved standard – seventh edition.Clinical and Laboratory Standards Institute, Wayne, PA. 10
ACCEPTED MANUSCRIPT 22.
Farrow KA, Lyras D, Rood JI. 2001. Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile. Microbiology 147:2717-2728.
23.
Lyras D, Rood JI. 1996. Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens. Antimicrob Agents Chemother
24.
RI PT
40:2500-2504.
Spigaglia P, Barbanti F, Mastrantonio P. 2008. Tetracycline resistance gene tet(W) in the pathogenic bacterium Clostridium difficile. Antimicrob Agents Chemother
25.
SC
52:770-773.
Wang H, Roberts AP, Lyras D, Rood JI, Wilks M, Mullany P. 2000.
M AN U
Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX. J Bacteriol 182:3775-3783. 26.
Dridi L, Tankovic J, Burghoffer B, Barbut F, Petit JC. 2002. gyrA and gyrB
TE D
mutations are implicated in cross-resistance to ciprofloxacin and moxifloxacin in Clostridium difficile. Antimicrob Agents Chemother 46:3418-3421. 27.
Cheng AC, Ferguson JK, Richards MJ, Robson JM, Gilbert GL, McGregor A,
EP
Roberts S, Korman TM, Riley TV, Australasian Society for Infectious Diseases.
AC C
2011. Australasian Society for Infectious Diseases guidelines for the diagnosis and treatment of Clostridium difficile infection. Med J Aust 194:353-358.
28.
Venuto C, Butler M, Ashley ED, Brown J. 2010. Alternative therapies for Clostridium difficile infections. Pharmacotherapy 30:1266-1278.
29.
Wullt M, Odenholt I. 2004. A double-blind randomized controlled trial of fusidic acid and metronidazole for treatment of an initial episode of Clostridium difficileassociated diarrhoea. J Antimicrob Chemother 54:211-216.
11
ACCEPTED MANUSCRIPT 30.
Young GP, Ward PB, Bayley N, Gordon D, Higgins G, Trapani JA, McDonald MI, Labrooy J, Hecker R. 1985. Antibiotic-associated colitis due to Clostridium difficile: double-blind comparison of vancomycin with bacitracin. Gastroenterology 89:1038-1045. Citron DM, Merriam CV, Tyrrell KL, Warren YA, Fernandez H, Goldstein EJ.
RI PT
31.
2003. In vitro activities of ramoplanin, teicoplanin, vancomycin, linezolid, bacitracin, and four other antimicrobials against intestinal anaerobic bacteria. Antimicrob Agents
32.
SC
Chemother 47:2334-2338.
Noren T, Akerlund T, Wullt M, Burman LG, Unemo M. 2007. Mutations in fusA
M AN U
associated with posttherapy fusidic acid resistance in Clostridium difficile. Antimicrob Agents Chemother 51:1840-1843. 33.
Slimings C, Riley TV. 2014. Antibiotics and hospital-acquired Clostridium difficile infection: update of systematic review and meta-analysis. J Antimicrob Chemother
34.
TE D
69:881-891.
Wasels F, Spigaglia P, Barbanti F, Mastrantonio P. 2013. Clostridium difficile erm(B)-containing elements and the burden on the in vitro fitness. J Med Microbiol
Bakker D, Corver J, Harmanus C, Goorhuis A, Keessen EC, Fawley WN, Wilcox
AC C
35.
EP
62:1461-1467.
MH, Kuijper EJ. 2010. Relatedness of human and animal Clostridium difficile PCR
ribotype 078 isolates determined on the basis of multilocus variable-number tandemrepeat analysis and tetracycline resistance. J Clin Microbiol 48:3744-3749.
36.
van Hoek AH, Mevius D, Guerra B, Mullany P, Roberts AP, Aarts HJ. 2011. Acquired antibiotic resistance genes: an overview. Front Microbiol 2:203.
37.
Spigaglia P, Barbanti F, Mastrantonio P, Brazier JS, Barbut F, Delmee M, Kuijper E, Poxton IR, European Study Group on Clostridium difficile. 2008. 12
ACCEPTED MANUSCRIPT Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe. J Med Microbiol 57:784-789. 38.
Slimings C, Armstrong P, Beckingham WD, Bull AL, Hall L, Kennedy KJ, Marquess J, McCann R, Menzies A, Mitchell BG, Richards MJ, Smollen PC,
RI PT
Tracey L, Wilkinson IJ, Wilson FL, Worth LJ, Riley TV. 2014. Increasing incidence of Clostridium difficile infection, Australia, 2011-2012. Med J Aust 200:272-276.
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Lim SK, Stuart RL, Mackin KE, Carter GP, Kotsanas D, Francis MJ, Easton M, Dimovski K, Elliott B, Riley TV, Hogg G, Paul E, Korman TM, Seemann T,
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Stinear TP, Lyras D, Jenkin GA. 2014. Emergence of a ribotype 244 strain of Clostridium difficile associated with severe disease and related to the epidemic
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ribotype 027 strain. Clin Infect Dis 58:1723-1730.
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39.
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ACCEPTED MANUSCRIPT Table 1. Toxinotyping data for clinical isolates of C. difficile from Melbourne, Victoria. toxinotype toxin
cdtB PCR
tcdC sequenceb no. of isolates, no. of isolates,
profilea
historic (%)
recent (%) 93 (75.6)
A+B+CDT-
-
WT
34 (68)
I
A+B+CDT-
-
WT
2 (4)
7 (5.7)
II
A+B+CDT-
-
WT
1 (2)
0
+
C184T; 39 bp 0
V
+
A B CDT
deletion A-B+CDT-
XIa
A-B-CDT+
+
WT
0
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VIII
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+ +
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0
C184T; 39 bp 2 (4)
5 (4.1)
2 (1.6) 1 (0.8)
deletion
A+B+CDT-
XII
-
XIV
+
A B CDT
4 (8)
C191A; 36 bp 0
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+ + +
WT
3 (2.4) 1 (0.8)
deletion
XXIV
A+B+CDT+ + -
- -
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non-
A B CDT
total
0
1 (0.8)
-
7 (14)
10 (8.1)
50 (100)
123 (100)
-
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toxigenic
WT
a
A = toxin A; B = toxin B; CDT = binary toxin; + = positive; - = negative;
b
WT = wild type (identical to the VPI10463 reference strain).
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ACCEPTED MANUSCRIPT Table 2. Antimicrobial susceptibility data for historic Victorian strains of C. difficile (n = 9). antimicrobial MIC50 (µg/ml)
MIC90 (µg/ml)
MIC range (µg/ml) resistance
agent a
no.
breakpoint resistant (µg/ml) b
(%)
n.a.
-
≥32
0 (0)
≥8
9 (100)
n.a.
-
n.a.
-
≥8
0 (0)
1–4
≥2
7 (78)
≤1 – 64
≥16
2 (22)
0.25-0.5
n.a.
-
>512
>512
>512
CHL
8
8
4–8
CLI
8
16
4 - >32
ERY
2
>32
1 - >32
FUS
1
4
1–8
MXF
1
1
1–2
PEN
2
2
TET
≤1
16
TEI
0.5
0.5
MTZ
0.5
0.5
≤0.25 – 1
≥32
0 (0)
VAN
1
2
0.5 – 2
≥2c
0 (0)
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a
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BAC
BAC, bacitracin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; FUS, fusidic
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acid; MXF, moxifloxacin; PEN, penicillin; TET, tetracycline; TEI, teicoplanin; MTZ,
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metronidazole; VAN, vancomycin. b
resistance breakpoints based on CLSI guidelines for anaerobes; n.a. = not available.
c
Vancomycin resistance breakpoint based on EUCAST guidelines.
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ACCEPTED MANUSCRIPT Table 3. Antimicrobial susceptibility data for recent Victorian strains of C. difficile (n =14). antimicrobial MIC50 (µg/ml)
MIC90 (µg/ml)
agent a
MIC range resistance (µg/ml)
no.
breakpoint resistant (µg/ml) b
(%)
>512
>512
512 - >512 n.a.
-
CHL
8
8
4–8
≥32
0 (0)
CLI
8
16
4 - >32
≥8
13 (93)
ERY
2
>32
2 - >32
n.a.
-
FUS
1
8
0.5 – 8
n.a.
-
MXF
1
16
0.5 - 16
≥8
2 (14)
PEN
2
4
1–4
≥2
11 (79)
TET
≤1
≤1
≤1 - 16
≥16
1 (7)
TEI
0.5
0.5
0.25-0.5
n.a.
-
MTZ
0.5
1
0.5 – 1
≥32
0 (0)
VAN
1
2
0.5 – 2
≥2c
0 (0)
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a
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BAC
BAC, bacitracin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; FUS, fusidic
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acid; MXF, moxifloxacin; PEN, penicillin; TET, tetracycline; TEI, teicoplanin; MTZ,
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metronidazole; VAN, vancomycin. b
resistance breakpoints based on CLSI guidelines for anaerobes; n.a. = not available.
c
Vancomycin resistance breakpoint based on EUCAST guidelines.
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S1075-9964(15)30017-2
DOI:
10.1016/j.anaerobe.2015.05.001
Reference:
YANAE 1441
To appear in:
Anaerobe
Received Date: 24 January 2015 Revised Date:
24 April 2015
Accepted Date: 1 May 2015
Please cite this article as: Mackin KE, Elliott B, Kotsanas D, Howden BP, Carter GP, Korman TM, Riley TV, Rood JI, Jenkin GA, Lyras D, Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia, Anaerobe (2015), doi: 10.1016/ j.anaerobe.2015.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia
Carter a, Tony M. Korman
c,f
, Thomas V. Riley
b,g
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Kate E. Mackin a, Briony Elliott b, Despina Kotsanas c, Benjamin P. Howden d,e, Glen P. , Julian I. Rood a, Grant A. Jenkin c,
a
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and Dena Lyras a*
Department of Microbiology, Monash University, Clayton, VIC, Australia,
b
School of
Australia, d
c
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Pathology and Laboratory Medicine, The University of Western Australia, Nedlands, WA, Monash Infectious Diseases, Monash Health, Clayton, VIC, Australia,
Department of Microbiology, Austin Health, Heidelberg, VIC, Australia, e Department of
Microbiology and Immunology, University of Melbourne, VIC, Australia, f Department of
Nedlands, WA, Australia.
g
PathWest Laboratory Medicine,
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Microbiology, Monash Health, Clayton, VIC, Australia,
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*corresponding author: [email protected] phone: +61 3 9902 9155
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postal address: Department of Microbiology, Building 76, Monash University, Victoria, Australia 3800
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ACCEPTED MANUSCRIPT ABSTRACT Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were
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susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only
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detected in recent strains.
HIGHLIGHTS:
C. difficile is a nosocomial pathogen, with different strain types causing disease.
•
We have generated a snap-shot of C. difficile strain types circulating in Victoria.
•
The diversity of isolates in Victoria may differ from other parts of the world.
•
Most strains were toxinotype 0, but toxinotypes V and VIII strains were also found.
•
Strains were metronidazole and vancomycin sensitive; 2 were moxifloxacin resistant.
BODY OF PAPER
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Clostridium difficile is the most commonly recognised cause of antibiotic-associated diarrhea in humans (1). The development of C. difficile infection (CDI) is linked to disruption of the
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host microbiota (2), which often occurs after antibiotic therapy. The major factors contributing to disease are toxins A and B (3). The genes encoding these toxins are found on a chromosomally-located region, the Pathogenicity Locus (PaLoc). Variations in the PaLoc may result in differences in the toxins produced or in their regulation. This variation is the basis of toxinotyping, which groups strains by different PCR-RFLP patterns in sections of the PaLoc (4). Some strains also produce a third toxin, CDT, which may act as a colonisation factor (5). Increased morbidity and mortality due to C. difficile worldwide over the last 2
ACCEPTED MANUSCRIPT decade has been linked to fluoroquinolone-resistant toxinotype III/ribotype 027 strains, which may be more virulent than other strain types (6, 7). The first case of locally-acquired ribotype 027 in Australia was identified in Melbourne, Victoria, in 2010 (8). In addition, suggestions that toxinotype V/ribotype 078 isolates may demonstrate increased virulence capacity (9, 10)
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are of concern, while toxinotype VIII/ribotype 017 isolates have also been associated with outbreaks of severe disease (11). The ability of strains to spread and persist in the healthcare setting means there is a need to monitor isolates for the emergence of these strain types in
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new locations. Antibiotic stewardship often plays a central role in infection control practices targeting C. difficile. Thus, knowledge of the strain types present in a given hospital and their
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antibiotic susceptibility profiles may be useful in targeting this organism. However, the only published study examining antibiotic resistance in Australia is from the state of New South Wales in 2002 (12), with no published research papers available regarding isolates from other
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parts of the country.
This study aimed to generate an understanding of the C. difficile strain types present in the state of Victoria at different times. Two healthcare networks contributed C. difficile isolates
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from patients (n = 123) during 2006-2009. In addition, 50 historic (1979-1989) C. difficile
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strains from Victoria were characterised. All strains were toxinotyped, to examine variation in the PaLoc (4), and non-toxigenic isolates were confirmed by PCR (13). PCR was used to screen isolates for the presence of cdtB (14), which encodes part of CDT, and the tcdC gene, encoding a toxin regulator, was sequenced (15) (Table 1). We found that 75.6% of recent and 68% of historic Victorian isolates were toxinotype 0, in line with previous studies in other countries where toxinotype 0 can comprise 75-80% of isolates (12, 16). Recent strains included toxinotypes V (5/123) and VIII (2/123); these types were not found in the older
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ACCEPTED MANUSCRIPT isolates of this study. No toxinotype III/ribotype 027 strains were identified in either time period.
Ribotyping analysis (17) revealed further diversity of strain types in Victoria, with
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approximately 31% of recent isolates belonging to ribotype 014; this type is often found elsewhere in the world (18, 19). The next most common ribotypes identified (6% each of recent isolates) were 054, which is not often found in other countries (18, 20), and a unique
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Australian ribotype with no major worldwide equivalent. This data suggests that the diversity of C. difficile clinical isolates in Victoria may differ from other parts of the world. Of the
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toxinotype V isolates, one each belonged to the closely-related ribotypes 126 and 127. Two other toxinotype V isolates were ribotype 078, confirming the presence of this strain type in Victoria.
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We also examined a selection of isolates for antimicrobial susceptibilities (n = 23). Nine of these isolates were historic strains while 14 were recent isolates (Tables 2 and 3). The antibiotics tested were bacitracin, clindamycin, chloramphenicol, fusidic acid, erythromycin,
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moxifloxacin, penicillin, tetracycline, teicoplanin, vancomycin and metronidazole. MICs
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were determined following Clinical and Laboratory Standards Institute (CLSI) guidelines for agar dilution, with resistance breakpoints based on CLSI recommendations for anaerobes (21) where available. The resistance breakpoint determined by The European Committee on Antimicrobial Susceptibility Testing (EUCAST) for vancomycin (>2 µg/ml) (clinical break points – bacteria v.3.0; http://www.eucast.org/clinical_breakpoints/) was used as there is no CLSI recommendation. Where possible, resistance mechanisms were identified by PCR and/or sequencing, using primers specific for erm(B) (22), tet(M) (23), tet(W) (24), tetA(P)
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ACCEPTED MANUSCRIPT (23), tetB(P) (23), tndX (25), int (5’ CAGGTCTTCGTATTTCAGAG 3’ and 5’ TGTATGTCGCAAACTATG 3’), gyrA (26) and gyrB (26).
In Australia, metronidazole is the recommended first-line treatment for non-severe CDI and
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vancomycin is recommended for severe cases and recurrent disease (27). All of the isolates tested in this study were susceptible to metronidazole and vancomycin. Alternatives suggested for the treatment of CDI include teicoplanin (28) fusidic acid (29), and bacitracin
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(30). All isolates tested in this study demonstrated low teicoplanin MICs. However, all showed high bacitracin MICs, and 35% of recent isolates had high MICs to fusidic acid.
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Resistance to both of these agents has been documented previously (31, 32), suggesting that these two antimicrobials may not be viable alternatives.
Clindamycin use is strongly associated with the development of CDI (33). All isolates tested,
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except one, were resistant to clindamycin. Five of these isolates also showed high erythromycin MICs. In C. difficile this resistance is often encoded by an erm(B) gene, which may be carried on a transposon (22, 34); three of these five isolates were positive for erm(B)
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by PCR. The other two isolates were negative in this PCR suggesting that another mechanism
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may be responsible for the reduced susceptibility of these strains. These isolates both demonstrated high erythromycin MICs (>32 µg/ml), but low-level clindamycin resistance (8 µg/ml).
One of the erm(B)-containing isolates demonstrated high MICs to many of the antimicrobial agents, showing resistance to fusidic acid, penicillin, and tetracycline. The tetracycline resistance determinant appears to be carried on Tn5397 with positive PCR results for both tet(M) and tndX, as the latter is specific to this element. Another isolate, a recent toxinotype 5
ACCEPTED MANUSCRIPT V/ribotype 078 strain, contains a Tn916-like element as indicated by positive PCR results for tet(M) and the Tn916-specific int gene; such elements have been found in this strain type previously (35). Tetracycline resistance in the Clostridia may be mediated by other tet genes
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(24, 36), however, these could not be detected in the third tetracycline-resistant isolate.
While the development of fluoroquinolone resistance has been associated with the spread of C. difficile ribotype 027 (5), it is seen in other strain types (37). In this study, decreased
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susceptibility to moxifloxacin (MIC = 16 µg/ml) was found in two recent isolates, while all of the historic isolates tested were fully susceptible. In C. difficile resistance to
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fluoroquinolones is mediated by mutations in the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB (26), with the most common change resulting in the substitution of threonine to isoleucine at position 82 of GyrA (37). This same substitution was found in
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the two isolates in this study.
The rates of C. difficile infection are increasing in Australia (38) and we have very limited information about the types of strains circulating in our population. While much of the focus
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has been on C. difficile ribotype 027, other strain types can cause outbreaks of disease, as
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recently seen in our local area (39). This study has identified the predominant C. difficile toxinotypes in two major Victorian healthcare networks during non-outbreak conditions, and demonstrated that these isolates may be resistant to many antimicrobial agents. Many of the isolates identified in this study were toxinotype 0, consistent with findings in other parts of the world. However, the results of this study are also consistent with the hypothesis that the epidemiology of C. difficile is different in Australia compared to other countries. Of particular concern is the identification of toxinotype V and toxinotype VIII strains in this study. 6
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The impact of variant C. difficile strain types abroad highlights why the prevention and control of this organism in Australian healthcare settings is of paramount importance. The recent move away from culturing for C. difficile towards other methods such as PCR or
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enzyme immunoassay means that important strain types cannot be detected if or when they do emerge in Australia, and antibiotic resistance properties cannot be determined. This is a concern not just in terms of imported strains posing a threat to Australian biosecurity, but also
ACKNOWLEDGMENTS
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in that uniquely Australian strain types could disseminate elsewhere.
The researchers were supported by Project Grant GNT1051584 from the Australian National
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Health and Medical Research Council (NHMRC) and Discovery Grant DP1093891 from the Australian Research Council (ARC). DL was supported by an ARC Future Fellowship (FT120100779) from the ARC. BH was supported by an NHMRC Career Development
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Fellowship (GNT1023526).
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ACCEPTED MANUSCRIPT REFERENCES
1.
McDonald LC, Owings M, Jernigan DB. 2006. Clostridium difficile infection in patients discharged from US short-stay hospitals, 1996-2003. Emerg Infect Dis
2.
RI PT
12:409-415.
Reeves AE, Theriot CM, Bergin IL, Huffnagle GB, Schloss PD, Young VB. 2011. The interplay between microbiome dynamics and pathogen dynamics in a murine
3.
SC
model of Clostridium difficile infection. Gut Microbes 2:145-158.
Carter GP, Awad MM, Kelly ML, Rood JI, Lyras D. 2011. TcdB or not TcdB: a
4.
M AN U
tale of two Clostridium difficile toxins. Future Microbiol 6:121-123. Rupnik M, Avesani V, Janc M, von Eichel-Streiber C, Delmee M. 1998. A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 36:2240-2247.
Schwan C, Stecher B, Tzivelekidis T, van Ham M, Rohde M, Hardt WD,
TE D
5.
Wehland J, Aktories K. 2009. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog
Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S, Bourgault
AC C
6.
EP
5:e1000626.
AM, Nguyen T, Frenette C, Kelly M, Vibien A, Brassard P, Fenn S, Dewar K, Hudson TJ, Horn R, Rene P, Monczak Y, Dascal A. 2005. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med 353:2442-2449.
7.
Rupnik M, Wilcox MH, Gerding DN. 2009. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7:526-536.
8
ACCEPTED MANUSCRIPT 8.
Richards M, Knox J, Elliott B, Mackin K, Lyras D, Waring LJ, Riley TV. 2011. Severe infection with Clostridium difficile PCR ribotype 027 acquired in Melbourne, Australia. Med J Aust 194:369-371.
9.
Goorhuis A, Bakker D, Corver J, Debast SB, Harmanus C, Notermans DW,
RI PT
Bergwerff AA, Dekker FW, Kuijper EJ. 2008. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis 47:1162-1170.
Knetsch CW, Hensgens MP, Harmanus C, van der Bijl MW, Savelkoul PH,
SC
10.
Kuijper EJ, Corver J, van Leeuwen HC. 2011. Genetic markers for Clostridium
11.
M AN U
difficile lineages linked to hypervirulence. Microbiology 157:3113-3123. Drudy D, Harnedy N, Fanning S, Hannan M, Kyne L. 2007. Emergence and control of fluoroquinolone-resistant, toxin A-negative, toxin B-positive Clostridium difficile. Infect Control Hosp Epidemiol 28:932-940.
Leroi MJ, Siarakas S, Gottlieb T. 2002. E test susceptibility testing of nosocomial
TE D
12.
Clostridium difficile isolates against metronidazole, vancomycin, fusidic acid and the
21:72-74.
Braun V, Hundsberger T, Leukel P, Sauerborn M, von Eichel-Streiber C. 1996.
AC C
13.
EP
novel agents moxifloxacin, gatifloxacin, and linezolid. Eur J Clin Microbiol Infect Dis
Definition of the single integration site of the pathogenicity locus in Clostridium
difficile. Gene 181:29-38.
14.
Stubbs S, Rupnik M, Gibert M, Brazier J, Duerden B, Popoff M. 2000.
Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile. FEMS Microbiol Lett 186:307-312.
9
ACCEPTED MANUSCRIPT 15.
Spigaglia P, Mastrantonio P. 2002. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol 40:3470-3475.
16.
Barbut F, Mastrantonio P, Delmee M, Brazier J, Kuijper E, Poxton I, European
RI PT
Study Group on Clostridium difficile. 2007. Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect 13:1048-1057.
Stubbs SL, Brazier JS, O'Neill GL, Duerden BI. 1999. PCR targeted to the 16S-
SC
17.
23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a
18.
M AN U
library consisting of 116 different PCR ribotypes. J Clin Microbiol 37:461-463. Bauer MP, Notermans DW, van Benthem BH, Brazier JS, Wilcox MH, Rupnik M, Monnet DL, van Dissel JT, Kuijper EJ, ECDIS Study Group. 2011. Clostridium difficile infection in Europe: a hospital-based survey. Lancet 377:63-73. Walk ST, Micic D, Jain R, Lo ES, Trivedi I, Liu EW, Almassalha LM, Ewing
TE D
19.
SA, Ring C, Galecki AT, Rogers MA, Washer L, Newton DW, Malani PN, Young VB, Aronoff DM. 2012. Clostridium difficile ribotype does not predict severe
Wilcox MH, Shetty N, Fawley WN, Shemko M, Coen P, Birtles A, Cairns M,
AC C
20.
EP
infection. Clin Infect Dis 55:1661-1668.
Curran MD, Dodgson KJ, Green SM, Hardy KJ, Hawkey PM, Magee JG, Sails AD, Wren MW. 2012. Changing epidemiology of Clostridium difficile infection
following the introduction of a national ribotyping-based surveillance scheme in England. Clin Infect Dis 55:1056-1063.
21.
Clinical and Laboratory Standards Institute. 2007. Methods for antimicrobial susceptibility testing of anaerobic bacteria; approved standard – seventh edition.Clinical and Laboratory Standards Institute, Wayne, PA. 10
ACCEPTED MANUSCRIPT 22.
Farrow KA, Lyras D, Rood JI. 2001. Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile. Microbiology 147:2717-2728.
23.
Lyras D, Rood JI. 1996. Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens. Antimicrob Agents Chemother
24.
RI PT
40:2500-2504.
Spigaglia P, Barbanti F, Mastrantonio P. 2008. Tetracycline resistance gene tet(W) in the pathogenic bacterium Clostridium difficile. Antimicrob Agents Chemother
25.
SC
52:770-773.
Wang H, Roberts AP, Lyras D, Rood JI, Wilks M, Mullany P. 2000.
M AN U
Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX. J Bacteriol 182:3775-3783. 26.
Dridi L, Tankovic J, Burghoffer B, Barbut F, Petit JC. 2002. gyrA and gyrB
TE D
mutations are implicated in cross-resistance to ciprofloxacin and moxifloxacin in Clostridium difficile. Antimicrob Agents Chemother 46:3418-3421. 27.
Cheng AC, Ferguson JK, Richards MJ, Robson JM, Gilbert GL, McGregor A,
EP
Roberts S, Korman TM, Riley TV, Australasian Society for Infectious Diseases.
AC C
2011. Australasian Society for Infectious Diseases guidelines for the diagnosis and treatment of Clostridium difficile infection. Med J Aust 194:353-358.
28.
Venuto C, Butler M, Ashley ED, Brown J. 2010. Alternative therapies for Clostridium difficile infections. Pharmacotherapy 30:1266-1278.
29.
Wullt M, Odenholt I. 2004. A double-blind randomized controlled trial of fusidic acid and metronidazole for treatment of an initial episode of Clostridium difficileassociated diarrhoea. J Antimicrob Chemother 54:211-216.
11
ACCEPTED MANUSCRIPT 30.
Young GP, Ward PB, Bayley N, Gordon D, Higgins G, Trapani JA, McDonald MI, Labrooy J, Hecker R. 1985. Antibiotic-associated colitis due to Clostridium difficile: double-blind comparison of vancomycin with bacitracin. Gastroenterology 89:1038-1045. Citron DM, Merriam CV, Tyrrell KL, Warren YA, Fernandez H, Goldstein EJ.
RI PT
31.
2003. In vitro activities of ramoplanin, teicoplanin, vancomycin, linezolid, bacitracin, and four other antimicrobials against intestinal anaerobic bacteria. Antimicrob Agents
32.
SC
Chemother 47:2334-2338.
Noren T, Akerlund T, Wullt M, Burman LG, Unemo M. 2007. Mutations in fusA
M AN U
associated with posttherapy fusidic acid resistance in Clostridium difficile. Antimicrob Agents Chemother 51:1840-1843. 33.
Slimings C, Riley TV. 2014. Antibiotics and hospital-acquired Clostridium difficile infection: update of systematic review and meta-analysis. J Antimicrob Chemother
34.
TE D
69:881-891.
Wasels F, Spigaglia P, Barbanti F, Mastrantonio P. 2013. Clostridium difficile erm(B)-containing elements and the burden on the in vitro fitness. J Med Microbiol
Bakker D, Corver J, Harmanus C, Goorhuis A, Keessen EC, Fawley WN, Wilcox
AC C
35.
EP
62:1461-1467.
MH, Kuijper EJ. 2010. Relatedness of human and animal Clostridium difficile PCR
ribotype 078 isolates determined on the basis of multilocus variable-number tandemrepeat analysis and tetracycline resistance. J Clin Microbiol 48:3744-3749.
36.
van Hoek AH, Mevius D, Guerra B, Mullany P, Roberts AP, Aarts HJ. 2011. Acquired antibiotic resistance genes: an overview. Front Microbiol 2:203.
37.
Spigaglia P, Barbanti F, Mastrantonio P, Brazier JS, Barbut F, Delmee M, Kuijper E, Poxton IR, European Study Group on Clostridium difficile. 2008. 12
ACCEPTED MANUSCRIPT Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe. J Med Microbiol 57:784-789. 38.
Slimings C, Armstrong P, Beckingham WD, Bull AL, Hall L, Kennedy KJ, Marquess J, McCann R, Menzies A, Mitchell BG, Richards MJ, Smollen PC,
RI PT
Tracey L, Wilkinson IJ, Wilson FL, Worth LJ, Riley TV. 2014. Increasing incidence of Clostridium difficile infection, Australia, 2011-2012. Med J Aust 200:272-276.
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Lim SK, Stuart RL, Mackin KE, Carter GP, Kotsanas D, Francis MJ, Easton M, Dimovski K, Elliott B, Riley TV, Hogg G, Paul E, Korman TM, Seemann T,
M AN U
Stinear TP, Lyras D, Jenkin GA. 2014. Emergence of a ribotype 244 strain of Clostridium difficile associated with severe disease and related to the epidemic
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ribotype 027 strain. Clin Infect Dis 58:1723-1730.
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39.
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ACCEPTED MANUSCRIPT Table 1. Toxinotyping data for clinical isolates of C. difficile from Melbourne, Victoria. toxinotype toxin
cdtB PCR
tcdC sequenceb no. of isolates, no. of isolates,
profilea
historic (%)
recent (%) 93 (75.6)
A+B+CDT-
-
WT
34 (68)
I
A+B+CDT-
-
WT
2 (4)
7 (5.7)
II
A+B+CDT-
-
WT
1 (2)
0
+
C184T; 39 bp 0
V
+
A B CDT
deletion A-B+CDT-
XIa
A-B-CDT+
+
WT
0
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VIII
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+ +
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0
C184T; 39 bp 2 (4)
5 (4.1)
2 (1.6) 1 (0.8)
deletion
A+B+CDT-
XII
-
XIV
+
A B CDT
4 (8)
C191A; 36 bp 0
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+ + +
WT
3 (2.4) 1 (0.8)
deletion
XXIV
A+B+CDT+ + -
- -
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non-
A B CDT
total
0
1 (0.8)
-
7 (14)
10 (8.1)
50 (100)
123 (100)
-
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toxigenic
WT
a
A = toxin A; B = toxin B; CDT = binary toxin; + = positive; - = negative;
b
WT = wild type (identical to the VPI10463 reference strain).
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ACCEPTED MANUSCRIPT Table 2. Antimicrobial susceptibility data for historic Victorian strains of C. difficile (n = 9). antimicrobial MIC50 (µg/ml)
MIC90 (µg/ml)
MIC range (µg/ml) resistance
agent a
no.
breakpoint resistant (µg/ml) b
(%)
n.a.
-
≥32
0 (0)
≥8
9 (100)
n.a.
-
n.a.
-
≥8
0 (0)
1–4
≥2
7 (78)
≤1 – 64
≥16
2 (22)
0.25-0.5
n.a.
-
>512
>512
>512
CHL
8
8
4–8
CLI
8
16
4 - >32
ERY
2
>32
1 - >32
FUS
1
4
1–8
MXF
1
1
1–2
PEN
2
2
TET
≤1
16
TEI
0.5
0.5
MTZ
0.5
0.5
≤0.25 – 1
≥32
0 (0)
VAN
1
2
0.5 – 2
≥2c
0 (0)
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a
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BAC
BAC, bacitracin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; FUS, fusidic
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acid; MXF, moxifloxacin; PEN, penicillin; TET, tetracycline; TEI, teicoplanin; MTZ,
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metronidazole; VAN, vancomycin. b
resistance breakpoints based on CLSI guidelines for anaerobes; n.a. = not available.
c
Vancomycin resistance breakpoint based on EUCAST guidelines.
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ACCEPTED MANUSCRIPT Table 3. Antimicrobial susceptibility data for recent Victorian strains of C. difficile (n =14). antimicrobial MIC50 (µg/ml)
MIC90 (µg/ml)
agent a
MIC range resistance (µg/ml)
no.
breakpoint resistant (µg/ml) b
(%)
>512
>512
512 - >512 n.a.
-
CHL
8
8
4–8
≥32
0 (0)
CLI
8
16
4 - >32
≥8
13 (93)
ERY
2
>32
2 - >32
n.a.
-
FUS
1
8
0.5 – 8
n.a.
-
MXF
1
16
0.5 - 16
≥8
2 (14)
PEN
2
4
1–4
≥2
11 (79)
TET
≤1
≤1
≤1 - 16
≥16
1 (7)
TEI
0.5
0.5
0.25-0.5
n.a.
-
MTZ
0.5
1
0.5 – 1
≥32
0 (0)
VAN
1
2
0.5 – 2
≥2c
0 (0)
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a
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BAC
BAC, bacitracin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; FUS, fusidic
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acid; MXF, moxifloxacin; PEN, penicillin; TET, tetracycline; TEI, teicoplanin; MTZ,
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metronidazole; VAN, vancomycin. b
resistance breakpoints based on CLSI guidelines for anaerobes; n.a. = not available.
c
Vancomycin resistance breakpoint based on EUCAST guidelines.
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