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Culture, identification and susceptibility testing of Clostridium difficile from EIA toxin positive faecal samples.

Introduction

Clostridium difficile is a spore-forming, obligate anaerobic, Gram positive bacillus. It is ubiquitous in the environment and causes disease in humans and animals. C. difficile infection is primarily a nosocomial infection associated with prolonged antibiotic use, particularly cephalosporins and/or fluoroquinolones. Genetically encoded toxins (commonly toxins A and/or B) are responsible for the in vivo effects of this organism. If undiagnosed or untreated, pseudomembranous colitis, toxic megacolon and death may result. C. difficile infection is treated with metronidazole and/or vancomycin (1).

Since the early 2000s, hypervirulent strains (particularly PCR ribotype 027, also known as NAP-1 or BI) have been responsible for major outbreaks and subsequent increased mortality in the northern hemisphere, especially North America and Europe (2-4). Overseas studies have suggested a link between high-level fluoroquinolone resistance and PCR ribotype (5,6). Hypervirulent strains have recently been detected in New Zealand (7,8).

At Canterbury Health Laboratories, detection of toxins A/B from faecal samples is performed by enzyme immunoassay (EIA) (Premier[TM] Toxins A&B, Meridian Bioscience). Daily batch testing ensures results are available within 24 hours. Culture, in comparison, can take several days. In addition, accurate identification by traditional methods is difficult as other Clostridium species can be hard to differentiate from C. difficile. Identification by molecular sequencing, while accurate, takes a minimum two days and is limited to specialist laboratories.

The aim of this study was to culture and identify C. difficile from EIA toxins A/B positive and equivocal faecal samples. Breakpoint susceptibilities were performed on isolates to determine a baseline for future monitoring of emerging antimicrobial resistance.

Methods

Faecal samples

All faecal samples which tested positive or equivocal for C. difficile toxins A/B during the period September 2008 to March 2010 were included in the study. A total of 154 faecal specimens (146 toxin positive and eight specimens with equivocal results) were stored at -20[degrees]C ([+ or -] 3[degrees]C).

C. difficile culture

Stored faecal samples were subsequently thawed, subjected to alcohol-shock treatment (9), cultured onto Cycloserine, Cefoxitin, Fructose (CCF) Agar (Fort Richard Laboratories, Auckland), and incubated anaerobically at 36[degrees]C ([+ or -] 1[degrees]C) for 48 hours ([+ or -] 4 hours). After incubation, plates were examined for typical, ragged edged colonies (Figure 1) then observed under ultra violet light. Colonies with characteristic chartreuse fluorescence were stored at -80[degrees]C ([+ or -] 3[degrees]C) in brain heart infusion broth supplemented with 15% glycerol.

C. difficile identification

Stored isolates were subsequently thawed and sub-cultured onto Columbia sheep blood agar (Fort Richard Laboratories, Auckland). Plates were incubated anaerobically at 36[degrees]C ([+ or -] 1[degrees]C) for 48 hours ([+ or -] 4 hours). Cultured isolates were identified using matrix-assisted laser desorption/ionisation time-of-flight (MALDI -TOF) mass spectrometry (Bruker Daltonics) using MALDI Biotype version 3.0 software. A small number of isolates were also identified by 16S rRNA DNA sequencing to confirm that MALDI-TOF identification was correct.

Susceptibility testing

Breakpoint susceptibilities were tested by CLSI agar dilution method (10). Ciprofloxacin (2 and 8 [micro]g/mL), vancomycin (8 and 32 [micro]g/mL), metronidazole (8 and 32 [micro]g/mL), amoxycilllin/ clavulanic acid (4/2 and 16/8 [micro]g/mL) and meropenem (4 and 16 [micro]g/mL), were incorporated into agar plates, prepared in-house using Oxoid Brucella agar base (Oxoid NZ Ltd) supplemented with 5ug hemin and 1ug vitamin [K.sub.1] per mL and 5% laked sheep blood. A sterilised replicator device with 32 x 3 mm pins was used to inoculate prepared plates with an inoculum density of 1 x [10.sup.5] CFU per 2.0 [micro]L spot. Plates of lower antimicrobial dilution were inoculated first. An inoculation control plate (Columbia sheep blood agar, Fort Richard Laboratories, Auckland) was inoculated between each antibiotic series and after all plates had been inoculated. C. difficile NCTC 11382 was used as the control strain. Results were interpreted by CLSI criteria, where available (10).

Results

Of the 154 samples cultured, 120 yielded bacterial growth with colonial morphology resembling C. difficile; four of these were from eight samples with EIA equivocal results. No growth was obtained from 28 samples. Six samples grew bacteria not resembling C. difficile. After the exclusion of duplicates, the remaining 112 isolates were stored at -80[degrees]C ([+ or -] 3[degrees]C). However, 11 of these were non-viable on sub-culture, leaving 101 isolates for further analysis.

MALDI-TOF identified 85/101 (84.2%) isolates as C. difficile, two as C. butyricum, one each as C. clostridioforme and C. symbiosium and three as non-clostridia. Nine isolates were unable to be identified by MALDI-TOF, but were subsequently identified by 16S rRNA sequencing; six as C. difficile, one C. neonatale, and two Clostridium spp., unable to be speciated. 16S rRNA sequencing confirmed the MALDI-TOF identification of a representative eight C. difficile isolates and the two C. butyricum. Neither the C. clostridioforme nor the C. symbiosium could be confirmed by sequencing.

A total of 91 confirmed C. difficile isolates were available for breakpoint susceptibility testing. Results are displayed in Table 1. One isolate grew at 2 [micro]g/mL ciprofloxacin, but was inhibited at 8 [micro]g/mL. All isolates grew on all inoculation control plates.

Discussion

Although culture and identification of C.difficile is not routinely performed, it is necessary for antimicrobial susceptibility monitoring and for ribotyping; both of which are important epidemiologically. Therefore, rapid and inexpensive, methods of culture and accurate identification are desirable.

Alcohol-shock eliminates non-spore forming gastro-enteric organisms and promotes spore production. When cultured onto selective, enrichment medium, such as CCF agar, spores germinate and a pure growth of C. difficile (if present) should result. 16S rRNA sequencing, the gold standard for organism identification, is specialised, time-consuming and costly. Colonial morphology and fluorescence are useful but other Clostridia mimic C. difficile, as shown in this study. For MALDI-TOF identification, organisms are taken from the anaerobic environment for only the short time taken to transfer them to the MALDI target. Therefore they remain viable, eliminating the need for repeated sub-culture if susceptibility testing and/or typing are required.

Culture and identification of C. difficile was confirmed for only 91 (59%) of 154 samples (62% of toxin A/B positive and 50% of equivocal samples). This is similar to the 57% recovery rate of Limbago et al (11), but lower than that achieved by Roberts et al (7). The failure to isolate C. difficile from toxin A/B positive samples could be attributed to a number of factors: suboptimal faecal storage conditions prior to culture, samples in which the organisms were no longer viable but toxin remained detectable, or cross reactivity of C. sordellii toxins with C. difficile toxins A/ B, which is a known limitation of the Premier[TM] Toxins A&B EIA procedure (12). Because C. sordellii does not fluoresce under ultra violet light, any isolates of this organism were not stored for further identification. Although no correlation between strength of EIA result and recovery of organisms was attempted, the lower isolation rate in EIA equivocal specimens suggests that organism recovery is more difficult in specimens with low numbers of toxigenic C. difficile. Furthermore, the failure of 11 isolates to survive -80[degrees]C ([+ or -] 3[degrees]C) storage h ad an impact on the confirmed culture rate. In the future, MALDI-TOF identification directly following isolation should eliminate the need for storage prior to analysis.

All isolates were susceptible to the antibiotics tested with the exception of ciprofloxacin. This is consistent with the findings of Roberts et al from other parts of New Zealand (7). The in vitro susceptibility of all isolates to metronidazole and vancomycin indicates that treatment with these agents is currently still valid in New Zealand. These isolates were not typed, but future surveillance to include PCR ribotyping, as well as susceptibility testing, is recommended.

In conclusion: alcohol-shock, combined with a selective, enrichment agar is an effective method of culture for C. difficile. MALDI-TOF is an inexpensive, simple and reliable tool for the identification of C. difficile. All isolates were susceptible to vancomycin, metronidazole, amoxicillin/clavulanic acid and meropenem, while most of the isolates appeared resistant to ciprofloxacin. However, close monitoring of antimicrobial susceptibility trends is important. PCR ribotyping of future isolates is recommended to detect possible emergence of hypervirulent strains.

Acknowledgements

I am grateful to the following colleagues at Canterbury Health Laboratories for their assistance and encouragement with this project: Trevor Anderson, Dr Mona Schousboe, Julie Creighton and David Beckingham.

Author information

Mary Stevens, QTA Dip Appl Sci, Medical Laboratory Technician

Canterbury Health Laboratories, Christchurch

mary.stevens@cdhb.health.nz

References

(1.) Ch eng AC, Ferguson JK, Richards MJ, Robson JM, Gilbert GL, McGregor A, et al. Australasian Society for Infectious Diseases guidelines for the diagnosis and treatment of Clostridium difficile infection. Med J Aust 2011; 194: 353-358.

(2.) Kuijper EJ, Coignard B, Tull P; ESCMID Study Group for Clostridium difficile; EU Member States; European Centre for Disease Prevention and Control. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 2006, 12(Suppl 6): 2-18.

(3.) Labbe A, Poirier, L, Maccannell D, Louie T, Savoie M, Beliveau C, et al. Clostridium difficile infections in a Canadian tertiary care hospital before and during a regional epidemic associated with the BI/NAP1/027 strain. Antimicrob Agents Chemother 2008; 52: 3180-3187.

(4.) Sundram F, Guyot A, Carboo I, Green S, Lilaonitkul M, Scourfield A. Clostridium difficile ribotypes 027 and 106: clinical outcomes and risk factors. J Hosp Infect 2009; 72: 111-118.

(5.) Bourgault AM, Lamothe F, Loo VG, Poirier L; CDAD-CSI Study Group. In vitro susceptibility of Clostridium difficile clinical isolates from a multi-institutional outbreak in Southern Quebec, Canada. Antimicrob Agents Chemother 2006; 50: 3473-3475.

(6.) Coia, JE. What is the role of antimicrobial resistance in the new epidemic of Clostridium difficile? Int J Antimicrob Agents 2009; 33(Suppl 1): S9-S12.

(7.) Roberts S, Heffernan H, Al Anbuky N, Pope C, Paviour S, Camp T, et al. Molecular epidemiology and susceptibility profiles of Clostridium difficile in New Zealand, 2009. N Z Med J 2011; 124: 45-51.

(8.) Roberts S, Heffernan H, Al Anbuky N, Richardson A, Swager T, Taylor SL, et al. Epidemic strains of Clostridium difficile are present in Auckland, New Zealand. N Z Med J 2011; 124: 97-101.

(9.) Health Protection Agency. 2008. Processing of faeces for Clostridium difficile National Standard Method BSOP 10 Issue 1.2. http://www/hpa-standarmethods.org.uk/pdf sops.asp.

(10.) Clinical and Laboratory Standards Institute. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard--Seventh edition 2007. CLSI document M11-A7, Clinical and Laboratory Standards Institute: Wayne, PA, USA.

(11.) Limbago BM, Long CM, Thompson AD, Killgore GE, Hannett GE, Havill NL, et al. Clostridium difficile strains from community-associated infections. J Clin Microbiol 2009; 47: 3004-3007.

(12.) Package insert, Premier[TM] Toxins A&B. Meridian Bioscience. Catalogue number 616096.

Table 1. Antimicrobial susceptiblity of C. difficile
isolates

Antimicrobial        MIC breakpoints      Susceptible
agent                 ([micro]g/mL)           (%)

                      S           R

Ciprofloxacin *   [less than   [greater     2 (2.2)
                   or equal    than or
                    to] 2       equal
                                to] 8

Vancomycin *      [less than   [greater    91 (100)
                   or equal    than or
                    to] 8       equal
                                to] 32

Metronidazole     [less than   [greater    91 (100)
([dagger])         or equal    than or
                    to] 8       equal
                                to] 32

Amoxycillin/      [less than   [greater    91 (100)
clavulanic         or equal    than or
acid ([dagger])    to] 4/2      equal
                               to] 16/8

Meropenem         [less than   [greater    91 (100)
([dagger])         or equal    than or
                    to] 4       equal
                                to] 32

* Breakpoints obtained from Bourgault et al (5).
([dagger]) CLSI breakpoints (10).
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Author:Stevens, Mary
Publication:New Zealand Journal of Medical Laboratory Science
Date:Apr 1, 2013
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