Örebro University
School of Medicine
Medicine, Advanced course Degree project, 15 ECTS January 2015
The effect of LifeClean
TMon different Clostridium difficile ribotype spores
A pilot laboratory study
_____________________________________________________________________________________________________ Version 2
Author: Anna Rosengren, Student Supervisor: Hans Fredlund, MD, PhD Laboratory technician supervisor: Karin Johansson, Medical laboratory scientist School of health and medicine,
Örebro University, Örebro, Sweden
Abstract
Introduction: Clostridium difficile (CD) is a spore forming anaerobic rod. It is the major cause of Clostridium difficile infection (CDI), ranging from mild to severe diarrhea with lethal complications. Over the last 10 years hospital CDI outbreaks have increased. This is associated with spread of more virulent CD strains. Since CD sporulates, it is difficult to decontaminate which makes the search for effective sporicidal properties important. Objective: This pilot laboratory study explores the sporicidal effect of LifeCleanTM on five different epidemic CD spores, ribotypes 012, 017, 027, 046 and 078.
Method: The five CD ribotypes were cultured and spore suspensions were made. The viability of the spores was tested by 10 min exposure to LifeCleanTM and ethanol (70%). Saline was used as control. The samples were cultured on blood-‐agar plates and the colonies counted.
Results: The data shows a significant reduction of all ribotype spores after treatment with both LifeClean (p≤0.001) and ethanol (p≤0.001) using water at control. Also the difference between ethanol and LifeCleanTM was significant (p≤0.005). No significant difference in spore reduction (p=0.079) could be observed between the various ribotype spores.
Conclusion: The study shows that LifeCleanTM is an effective decontaminant with sporicidal effects on all CD ribotype spores tested. No differential sporicidal effect could be observed against the various subtype spores. Thus, LifeCleanTM is probably well suited to be used as a disinfectant in the clinical setting.
Abbreviations
CD – Clostridium difficile
CDI – Clostridium difficile infection tcdA – Toxin A
tcdB – Toxin B
PaLoc – CD pathogenicity locus tcdC – regulatory gene
CDT – binary toxin
ECDC– European Centre for Disease Prevention and Control FAAAP – Fastidious Anaerobe Agar Plates
CDAGP – Clostridium Difficile Agar Plates
CONTENTS
1. INTRODUCTION 5
1.1 Clostridium difficile 5
1.2 Clostridium difficile Infection 5
1.3 Virulence factors 5
1.4 Sporulation and spores 6
1.5 Clostridium difficile strains 6 1.6 Clostridium difficile disinfectants 7
2. OBJECTIVE 7
2.1 Aim 7
2.2 Hypothesis 7
3. METHOD AND MATERIAL 7
3.1 Clostridium difficile strains 7
3.2 Test solution 8
3.3 Agar plates 8
3.4 Filter 8
3.5 Spore suspension preparation 8
3.6 Spore concentration determination 9
3.7 Spore viability 9
3.8 Ethics 10
3.9 Calculations and Statistics 10
4. RESULTS 11 5. DISCUSSION 13 6. CONCLUSION 15 7. ACKNOWLEDGEMENTS 15 8. REFERENCES 16
1. INTRODUCTION 1.1 Clostridium difficile
Clostridium difficile (CD) is an anaerobic Gram-‐positive rod that tends to sporulate when environment is unfavourable. It is one of the most common hospital-‐associated pathogens [1]. CD strains have different characteristics and only some of them produce toxins causing “Clostridium difficile-‐associated diarrhea” or Clostridium difficile
infection (CDI) [2]. CD is known to spread nosocomially via the oral-‐fecal route and can cause hospital outbreaks [3]. CD may also spread among healthy individuals in a
community setting [4]. When a CD spore is ingested the environment is once again favourable and the spore germinates. CD is present in the stools of about 4-‐15% of the general population [4]. Vegetative CD cell can colonise the colon, but in individuals with normal gut-‐microflora the consequences are usually few, if any. This is probably due to other normal flora inhibiting the CD growth. However, if the gut flora balance is
disturbed CD can grow dominant and cause CDI.
1.2 Clostridium difficile infection
Various types of antibiotics are the main cause of disturbed the gut flora causing CDI, but clindamycin, cephalosporins and fluoroquinolones are considered the major risk
factors [5]. Other main risk factors are old age (>65 years), hospitalization and
comorbidities [6]. Of all hospital patients with antibiotic associated diarrhea CD causes approximately 25% [7]. CDI symptoms can range from mild to severe diarrhea, with lethal complications as pseudomembranous colitis, toxic megacolon and possible intestinal perforation [8]. Over the last 10 years hospital CDI severity and outbreaks have increased. This epidemiological change is considered to be associated with the occurrence and spread of more virulent CD strains, especially PCR ribotype 027 [9]. Also, in recent years more CD strains have developed resistance to various types of antibiotics [10].
1.3 Virulence factors
The major CD virulence factors are two polypeptide toxins, toxin A and toxin B. The genes encoding these toxins (tcdA and tcdB) are on the CD pathogenicity locus (PaLoc), together with two regulatory genes (tcdC and tcdR) and a gene (tcdE) proposed to take part in the toxins release [11]. Toxin A and B have a similar structures and both damage
cytoskeletal structure, cause cell death, disruption of tight junctions, followed by altered epithelial permeability, fluid secretion and inflammatory response [12]. There are other virulence factors encoded outside the PaLoc, as a binary toxin (CDT) and S-‐layer
proteins surrounding the CD (cell wall-‐associated proteins), which vary among strains and contributes to the CD pathogenicity in different degrees [13].
1.4 Sporulation and spores
The signals triggering CD sporulation are not fully understood, but are presumably related to environmental stimuli. The CD spore structure and morphology can be
compared to that of spores from other endospore-‐forming bacteria [14]. The first step in creating an endospore is the formation of a polar septum, which produces a small
forespore and a large mother cell. The mother cell engulfs the forespore and produces the cortex, coat and exosporium around the spore. During the mother cell lysis the spore is released. An endospore is dormant and resistant to harsh environments. This allows the CD spore to survive on different environmental surfaces up to 5 months. As a result there is a greater risk for patients to be infected if placed in a room of a prior CD infected occupant [15].
1.5 Clostridium difficile strains
Across Europe there is a great diversity among CD strains. In total, the prevalence of ribotype 027 more than tripled during the recent five-‐year period 2008-‐2013 [16]. The increased virulence of ribotype 027 may be caused by a frameshift mutation in tcdC, yielding high toxin levels, but other factors may additionally affect toxin production and virulence [17,18]. In a European survey ribotype 078 appeared as the third most
prevalent in Europe (2008). Ribotype 078 has been described to have similar increased virulence as 027, but data are conflicting [19]. In a Swedish national survey (2008) the majority of the isolates (78 %) belonged to four ribotypes, among them, 012, 017 and 046, were epidemic strains with decreased antibiotic susceptibility, hence prone to nosocomial disease spread [20]. The same study showed that ribotype 027 was
uncommon in Sweden. Ribotype 046 and 078 are also associated with animal infections which are possible routs of transmissions needed to be further studied [21,22]. These five ribotypes are not only found in Sweden and Europe but probably also in many other countries worldwide. They are epidemic and considered problematic because of their
more virulent spread. The five ribotypes were thus chosen to analyse in the present study.
Clostridium difficile disinfectants
Because of the endurance of CD spores many common cleaning agents such as ethanol are ineffective since they lack sporicidal capacity [23]. It is known that contaminated hands of healthcare personal and surfaces are sources of CD transmission in hospitals [24]. Hence, there is an urgent need of sporicidal detergents. Chlorine releasing
substances such as hypochlorite are known to be very effective against spores, but are disliked due to unpleasant odour, corrosive and irritant properties [24]. The commercial agent LifeCleanTM (LifeClean International AB, Uddevalla, Sweden) is a chlorine dioxide containing solution that is less corrosive and irritant and claim sporicidal properties. The present pilot laboratory study will further explore this sporicidal effect on different ribotype spores, which no previously study that could be found has done.
2. OBJECTIVE 2.1 Aim
The objective of this pilot laboratory study is to explore the sporicidal effect of
LifeCleanTM on five different spores of epidemic CD ribotypes (012, 017, 027, 046 and 078).
2.2 Hypothesis
LifeCleaneTM has a differential sporicidal capacity on CD ribotype spores.
3. METHOD AND MATERIAL 3.1 Clostridium difficile strains
Five strains were selected for this study: 012, 017, 027, 046 and 078. All strains are named according to the Cardiff ECDC collection [25]. The CD ribotypes used were received from the European Centre for Disease Prevention and Control (ECDC). All isolates were taken from patients with diarrheal disease. The samples were stored in -‐70 °C until analysed.
3.2 Test solution
In the present study, undiluted LifeCleanTM (1600 ppm) and ethanol (70%) were used as test solutions. Saline (aqueous solution of 0.85 % NaCl) was used as control.
3.3 Agar plates
In this study Fastidious Anaerobe Agar Plates (FAAAP) (5 % Horse Blood, defibrinated (SVA, Uppsala, Sweden), 4.6 % Fastidious Anaerobe Agar [50 % Peptone mixture, 11 % Sodium Chloride, 2 % Soluble Starch, 0.8 % Sodium Bicarbonate, 2 % Glucose, 2 % Sodium Pyruvate, 1 % L-‐Cysteine, 0.5 % Sodium Pyrophosphate, 2 % L-‐Arginine, 1% Sodium Succinate, 0.02 % Hemin, 0.002 % Vitamin K, 26% Agar] (Acumedia, Neogen Corp., Lansing, USA) and 0.1 % Taurocholate in 100 ml RO-‐filtered H2O were used. As control plates, selective Clostridium Difficile Agar Plates (4,57 % Fastidious Anaerobe Agar (Acumedia, Neogen Corp., Lansing, USA), 5 % Horse Blood, defibrinated (SVA, Sweden) and Clostridium Difficile Supplement (CDAGP) [500 mg/L D-‐ Cycloserine, 16 mg/L Cefoxitin] (Bergman Labora AB, Danderyd, Sverige) in 100 mL H2O) were used.
3.4 Filter
S-‐Pak filters were used (47 mm diameter, 0.45 μl pore size, white gridded; Merc Millipore; Billerica, MA, USA).
3.5 Spore suspension preparation
Each strain was applied on two FAAAPs. The plates were kept in an anaerobic incubator for 2 days at 36°C and then at room temperature for one week. A cotton swab was used to lift colonies from the two FAAAP surfaces and the colonies were submerged into 1.5
ml RO-‐filtered H2O. To eliminate remaining vegetative cells, 99.5 % ethanol was added
to a final concentration of 70 % and the samples were then thoroughly mixed and placed in a refrigerator (+4°C) for 1-‐2 h. Subsequently, the samples were centrifuged (+4°C) for 20 min at 3000 rpm. The supernatants were discarded and the pellets were mixed in 1
ml H2O. The two last steps were repeated four times resulting in final suspensions of
spores of the various strains.
3.6 Spore concentration determination
To determine spore concentrations in the various suspensions a 0.05 mm Bürker chamber was filled with 1/10 dilutions of the suspensions. Using a microscope (Eclips 50i, Nikon Instruments Europe BV, Amsterdam, Netherlands) the number of spores were counted and the concentrations calculated.
3.7 Spore viability
The viability of the five ribotype spores (012, 017, 027, 046 and 078) was tested by exposure to the disinfectant LifeCleanTM, to ethanol (70%) and as control to saline. 0.1 ml of each spore suspension was applied to a microscope slide. When dried, 0.2 ml of the test solution or controls were added for 10 min. Then the glass slide was placed in a sterile 500 ml glass bottle with 250 ml saline and put on a gyratory shaker (Gyrotory shaker model G2; New Brunswick scientific co. Inc., Edison, New Jersey, USA) at 200 rpm for 20 min. The resulting solution was treated in the following two ways:
Procedure I. Three samples (0.1 ml) of the solution, undiluted, diluted 1/10 and 1/100 were taken and spread across the surface of the FAAPs.
Procedure II. Two samples (1 ml and the remaining volume) of the solution were filtered. The filters were rinsed with 100 ml Peton-‐H2O (0.35 % KH2PO4, 0.71 %
Na2HPO42H2O, 0.42 % NaCl, 0.14 % Peptone Bacto). The filters were then applied to the surface of FAAAPs.
The resulting filtrates were treated in the following way: Samples of 40 ml were centrifuged, the supernatants discarded and the pellets resuspended in 0.1 ml saline. The suspensions were spread across the surface of CDAGPs.
To measure the remaining viable spores on the glass slides they were wiped off with a humidified cotton swab, which subsequently was inoculated in 2 ml saline. Three samples (0.1 ml) of the solution, undiluted, diluted 1/100 and 1/10.000 were taken and
spread across the surface of the FAAPs (Procedure III).
All the ager plates were incubated in anaerobic conditions for 2 days at 36°C and then
the growing colonies were counted, assuming that each colony represented one viable
spore.
3.8 Ethics
The CD strains were taken from infected patients, but since the patient's identity was not known it does not intrude on their privacy. There was a potential risk that the investigator or other personnel incurred infection, but carful hygiene procedures were used and the risk of infection was regarded as minimal and therefore acceptable.
3.9 Calculations and Statistics
The colonies were counted and recalculated to Colony-‐forming unit per ml (CFU/ml). From each of the three methods the most representative dilution from the dilution series was chosen. If a sample value was lacking due to overgrowth the next value of the following dilution was used, or if missing the value representing overgrowth (300 CFU) was chosen (see table 1, panel a).
The ratio between the water treated samples (mean value) and the original spore
solution was used to calculate the recoveries of the three methods. Since the recovery of the three methods varied, to be able to compare the different spore counts, the water treated samples were used as a reference resulting in a ratio between the LifeCleanTM treated and the water treated sample (see table 1, panel b). The adjusted values were used to calculate poled mean and median value (see table 1, panel c).
To calculate the significance of the differences between water, ethanol and LifeClean treated samples, Wilcoxon signed rank test was used. To analyse the significance of LifeCleans effects on the different ribotype spores, the Kruskal-‐Wallis test was used. Statistical analysis was performed using the IBM SPSS software package (version 22).
4. RESULTS
The results are shown in table 1. The recoveries of the three procedure were: 8.9, 1.2 and 5.2 %, respectively. The CDAGP controls were all negative.
Table 1: This table shows the overall results.
Table 1.
Panel a Panel b Panel c
Ribotype Sample P1 (CFU/ml) P2 (CFU/ml) P3 (CFU/ml) P1 ratio P2 ratio P3 ratio Mean ratio Median ratio O12 LC 0 5 000 13 000 0,0 0,0067 0,015 0,0072 0,0067 eth 0 30 000 850 000 0,0 0,040 1,0 0,35 0,040 H2O 4 400 000 750 000 850 000 1,0 1,0 1,0 1,0 1,0 O17 LC 13 000 0 3 000 0,0019 0,0 0,001 0,001 0,001 eth 380 000 160 000 1 300 000 0,056 0,21 0,38 0,21 0,21 H2O 6 700 000 750 000 3 300 000 1,0 1,0 1,0 1,0 1,0 O27 LC 25 000 14 000 30 000 0,0033 0,018 0,026 0,016 0,018 eth 300 000 370 000 2 600 000 0,040 0,50 2,3 0,93 0,50 H2O 7 500 000 750 000 1 200 000 1,0 1,0 1,0 1,0 1,0 O46 LC 0 0 4 300 0,0 0,0 0,002 0,001 0,0 eth 130 000 90 000 1 000 000 0,19 0,12 0,40 0,23 0,19 H2O 660 000 750 000 2 600 000 1,0 1,0 1,0 1,0 1,0 O78 LC 0 5 000 1 000 0,0 0,007 0,0014 0,003 0,001 eth 0 25 000 7 600 000 0,0 0,033 1,0 0,4 0,033 H2O 10 000 000 750 000 7 400 000 1,0 1,0 1,0 1,0 1,0
P1: Procedure I, P2: Procedure II and P3: Procedure III. CFU: Colonising forming units. LC: LifeCleanTM, eth: Ethanol 70%. H2O: Water control. Ratio: ratio between tests and water controls.
Fig 1. This figure shows the median ratios (a) and the log10 of the median ratios (b) of the samples in the various groups. The water sample (H2O) is used as reference (value 1). The difference between LifeClean treated samples and water controls were
significant; *: p≤0.001 (Wilcoxon signed rank test). Similarly, the difference between ethanol and water was significant; †: p≤0.001. Also the difference between ethanol and LifeCleanTM was significant; ‡: p≤0.005.
Fig 2: The mean ratio of LifeCleanTM treated samples (i.e. mean fractions) as well as standard deviations are shown in this figure. The distribution of the mean fractions is not significantly affected by LifeClean (p=0.079; Kruskal-‐Wallis test).
-‐4 -‐3,5 -‐3 -‐2,5 -‐2 -‐1,5 -‐1 -‐0,5 0 Fig 1b log10 median ratio
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04
O12 O17 O27 O46 O78 Fig 2 Mean fraction
0 0,2 0,4 0,6 0,8 1 1,2 Fig 1a median ratio
*
† ‡5. DISCUSSION
The present study set out to explore the sporicidal effect of LifeCleanTM on different CD ribotype spores (012, 017, 027, 046 and 078). The resulting data showed that there was a significant difference in spore reduction between the water treated and LifeCleanTM treated samples of all ribotype spores. Also, LifeCleanTM was much more efficient than ethanol. However, no significant deferential effect of LifeCleanTM on the various spores could be shown. Thus, LifeCleanTM is sporicidal and possibly well suited to
decontaminate these spores from CDI patients, but according to this study without any certain differential effect on the various ribotypes tested.
The method used was a CD spore carrier test designed at Örebro University Hospital modified according to AOAC (association of official agricultural chemists) [26]. This test was developed due to the absence of Clostridium difficile sporicidal standards and makes comparison between different studies possible [27]. The numbers of dilutions in the dilution series were few (2 or 3), and since one dilution generally was missing due to overgrowth or lack of sensitivity only one or two remained. Thus, it was not possible to fit any equations to the resulting values of the dilutions. Consequently, it is not known if the curves describing the dilution series were linear (y=ax + b), polynomial (y=ax2 + bx + c), exponential (y=ex) or other, making comparisons with studies by others difficult. In this study, a linear relation was presumed which however may be false and might cause the ratios between levels in LifeClean and water treated samples to be underestimated. Since the resulting data cannot be considered continuous but rather ordinal, non-‐ parametric statistical testing was used ensuring statistical calculations to be correct.
Locking at individual values it is obvious that one of the readings concerning the ethanol ribotype 027 sample is an outlier as is observed in table 1. However, this does not affect the statistical testing between water and LifeClean treated samples. This outlier could be due to coincidence or experimental failure such as improper sample dilution.
Although the statistical testing could not show any significant differences between the effects on the various ribotype spores, it should be noted that there was a trend towards difference that just fell short from significance (p=0.079; Kruskal-‐Wallis test). Thus, it is possible that the effect of LifeCleanTM on the various spores could differ. The data depicted in figure 2 might suggest that ribotype spores 017 and 046 are more sensitive
to LifeClean. To further examine this question new tests with a few modifications in the procedure could be performed: Particularly, for each ribotype spore the number of samples should be increased, and changing the dilution series would give more accurate results, both which would allow better statistical comparisons.
Furthermore, LifeCleanTM 1600 ppm was tested at one concentration only and results may differ at other concentrations. It was found by Goldenberg et al. that chlorine dioxide concentration at 1000 ppm was ineffective to decontaminate CD spores [28], whereas the present study showed significant CD spore reduction at 1600ppm concentration. This discrepancy emphasises the importance of the chlorine dioxide concentration used in relation sporicidal capacity. Also, higher chlorine dioxide concentration has been shown to be more effective on non-‐CD spores [29].
Dawson et al. found that other, non-‐chlorine dioxide based, disinfectants showed diverse effects on the ribotype spores 012, 017 and 027. The disinfectants properties fell in three categories; those dependent on only concentration, those dependent on only spore ribotype and those dependent on both spore ribotype and concentration [30]. In
accordance with Dawson’s finding it could be argued, that disinfectants also may affect the ribotype spores 046 and 078 differently. This would seem to strengthen the notion that the sporicidal effect of LifeCleaneTM may differ on the five ribotypes spores used in this study.
CD spores have commonly been found in CDI patients’ rooms. Most frequently on the patient’s bedside table, bedrail, patient-‐helper trapeze, patient’s call button and in the patient’s bathroom as well as toilet seat [28,31]. A European estimate year 2010 showed that hospitals additional costs per CDI related case ranged from 50,000 to 100,000 SEK causing society a substantial burden [32]. This emphasizes the urgent need of effective decontaminants to minimise CD spore transmission in hospitals. Speight et al. showed that eight of 19 different products with chlorine dioxide base was effective
decontaminators, achieving reduction in CD spore viability >103-‐fold using a contact time of 1 min. The remaining ten products were effective at 60 min. Five of these latter products were diluted more than the others possibly explaining the difference [33]. Thus, in agreement with the present study, these data from Speight et al. shows that products with chlorine dioxide base have a clinical relevance as decontaminants.
Since epidemic CD strains are nosocomially spread and can cause hospital outbreaks, it is important to know which detergents actually work on the various ribotypes. In the year 2011 an outbreak caused by ribotype 046 occurred at the hospital
Höglandssjukhuset and the adjacent area in the county of Jönköping (Sweden). In order to decontaminate wards and contaminated surfaces a non-‐sporicidal agent was used. Contrary to what was expected, infection rates increased. It was first in 2012 when hypochlorite was used that a 50 % reduction in infection rate could be observed [34]. Thus, detergents that eliminate all ribotypes, particularly epidemic strains, will decrease infection rates efficiently. Since hypochlorite is user and environmental unfriendly a disinfecting product such as LifeCleanTM would be preferable. Particularly if it could be shown that LifeCleanTM has a substantial effect on epidemic ribotypes.
Future studies are needed to further explore the sporicidal capacity of LifeCleanTM on different CD ribotype spores. In the present pilot study the results essentially are expressed as ordinal data and not continuous data due to insufficient number of
dilutions in the dilution series and thus not allowing proper ratio calculations. A future study in the hands of this author should include more extensive dilutions series or even better standard curves based on dilutions of the original CD spore solutions, in order to obtain results as continuous variables. Also, an increase number of samples would be necessary to increase the power of the statistical testing.
6. CONCLUSION
The results show that LifeCleanTM is an effective decontaminant with sporicidal effects on all CD ribotype spores tested. No differential sporicidal effect could be observed against the various subtype spores but possibly future studies will be able to do this. Thus, LifeCleanTM is probably well suited to be used as a disinfectant in the clinical setting.
7. ACKNOWLEDGEMENTS
I would like to thank my two supervisors Hans Fredlund and Karin Johansson for giving me the opportunity and help to perform this study. I would also like to thank Lars Rosengren, whose assistance with the statistics was most helpful, and to Linnea Hartman whose company I could not have been without the days at the laboratory.
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