THE PREVALENCE OF CLOSTRIDIUM DIFFCILE AT AIREDALE NHST ENVIRONMENT

THE PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AIREDALE NHST HOSPITAL ENVIRONMENT.
1
University Of Central Lancashire
School Of Pharmacy And
Biomedical Sciences
Bsc (Hons) Biomedical Science
(Part-Time)
Research Project Report
BL3296
The Prevalence of Clostridium Difficile at Airedale NHST
Hospital Environment.
Willard Erasmas Dzinyemba
G20269064
November, 2013

2
AUTHORSHIP DECLARATION
I, Willard E. Dzinyemba confirm that this dissertation and the work presented in it are my
own achievement.
Where I have consulted published work of others, or quoted from their work, this is attributed
and the source given. With these exceptions the entire dissertation is my own work;
I have acknowledged all main sources of help and contributors to relevant previous and on-
going research projects made in this area of research.
I also confirm that I have obtained informed consent from all people I have involved
in the work in this dissertation following the School's ethical guidelines.
I have read and understood the penalties associated with Academic Misconduct.
Signed:
Date: 22nd November, 2013

3
TABLE OF CONTENTS
1. Abstract
2. Introduction
2.1 Pathogenesis
2.2 Sources of Infection
2.3 Infection Prevention
3. Methods and Materials
3.1 Air Sampling
3.2 Environmental Surface Sampling
3.3 Soil and Cow dung
3.4 Pilot Study
3.5 Sampling and Processing Method Verification
3.6 Colony identification of Clostridium difficile
4. Results
5. Discussion
6. Conclusion
7. Sources of materials and manufacturers
8. Acknowledgements
9. References
10. Appendices

4
1. ABSTRACT
Clostridium difficile (CD) is a normal commensal bacterium of the adult gastrointestinal tract
which under certain conditions induces diseases like pseudomembranous colitis. It is a
nosocomial pathogen that is transmissible between patients in a hospital or from exogenous
sources to patients. There are variations in the reported prevalence of CD in hospital and
domestic environments. Most of the variations are due to the differences in sensitivities and
specificities of the methods used to isolate CD. The aim of this study was to determine the
prevalence and extent of Clostridium difficile (CD) contamination in ward and hospital
environments at Airedale General Hospital and from the farms that surround it using the
methods available at the hospital laboratory.
Air samples from the ward and hospital corridors were collected and tested. Premoistened
swabs were used to collect samples from ward surfaces around known Clostridium difficile
infected (CDI) patients, in corridors and farm cattle stoles. Soil samples were collected from
the hospital grounds and farms around the hospital. Cow dung was also collected from the
farms as it forms part of the hospital environment, and tested for CD.
Out of a total of 171 samples, CD was isolated from 3 (1.75%) samples. One (5.26%) of the
19 air samples was positive for CD and 2 (2.35%) out of 85 swabs collected were CD
positive. One isolate was non-toxigenic and awaiting PCR ribotype results and two isolates
were toxigenic by C. difficile GDH testing and Polymerase Chain Reaction (PCR) ribotype
027, and PCR ribotype 002 (table 4). No Clostridium difficile was isolated from the soil and
cow dung samples from the farms, air and surfaces near CDI patients except the floor (2).
Isolation of CD from air samples in hospital corridors show the sporadic contamination of air
away from symptomatic CDI patients which may be an exogenous source of CD.

5
The findings from this study also imply that adherence of health workers to infection
prevention protocols mainly hand hygiene. The cleaning detergent used at this hospital may
indicate that it is an effective sporicide and bactericide as shown by 0% of CD isolated from
all other sites especially contact areas near a patient with Clostridium difficile infection (CDI)
except from the floor. However, the repeated isolation of CD from the floor of this ward puts
in question the thoroughness of cleaning. The results also indicate that soil and cow dung in
their (hospital) environment do not pose a potential risk for exogenous CD transmission to
patients.
Adequate, more frequent and thorough decontamination of rooms and corridors may be
needed to minimise the risk of nosocomial infection with CD.
2. INTRODUCTION
Clostridium difficile infection is the most common cause of nosocomial diarrhoea with risk
factors which include advanced age, severity of underlying illness, gastrointestinal surgery,
the use of electronic rectal thermometers, and prior use of antimicrobials (Mayfield et al.
2000, Bartlett 1994, Loo et al. 2011, Koss et al. 2006, Friedman et al. 2013). It is associated
with mild diarrhoea, pseudomembranous colitis, and toxic megacolon (Bartlett 2008, Murray
et al. 2003, Yakob et al. 2013a). The infection results in an increased length of stay in
hospital ranging from 8 to 21 days which increases the cost of healthcare (Barbut et al. 2001,
Yakob et al. 2013a). It can cause sepsis and even death (McDonald et al. 2007, Muto et al.
2007). It has been isolated from healthy adults, asymptomatic neonates, animals, water from
rivers, lakes, sea and tap water, and also from soil (Malamou-Ladas et al. 1983, Al Saif et al.
1996). The infection is generally acquired nosocomially (Al Saif et al. 1996).
The reported prevalence rates by different studies were between 2% to 12% (Best et al.
2010), 7% - 13% (Martirosian 2006, Al Saif et al. 1996) Variations in the techniques used

6
account for these differences. Some studies used direct plating (Al Saif et al. 1996) and others
used sample enrichment methods (Akhi et al. 2011, Martirosian 2006, Vaishnavi et al.
2012). In this study, currently available techniques to the hospital laboratory for the isolation
of CD (direct plating) were used. A more preferred highly recommended and sensitive
method of enrichment using brain heart infusion with 1% sodium taurocholate (Akhi et al.
2011)was not used in this study due to budgetary constraints.
Clostridium difficile infection is a burden to health care facilities and Airedale NHS
foundation trust is no exception. Healthcare facilities have to deal with high financial costs of
morbidity and mortality related to CDI (Hill et al. 2013, Yakob et al. 2013a). While Figure 1
below shows a marked reduction of cases (25%) between 2011 and 2012 in England, Wales
and Northern Ireland, more measures need to be put in place to eradicate it and meet targets
(HPA 2012).
Figure 1: Data from (HPA 2012).

7
The number of cases of CD as apportioned by the department of health for Airedale NHS
trust for 2012 was 12. A total of 9 of the 12 were reported by October 2012 indicating high
incidence (Charlesworth 2012b).
Clostridium difficile was first isolated in 1935 from stool samples of new-born children and
named Bacillus difficilis (Lyerly et al. 1998). It was found to exist as a commensal organism
of the digestive tract of young infants (Bartlett 2008, Barbut et al. 2001, Lyerly et al. 1998).
Its toxins were also identified by (Bartlett 2008) as the cause of pseudomembranous colitis
for the first time in 1978 (George et al. 1979, Tenover et al. 2011, Bartlett 2008).
Clostridium difficile belongs to the family Clostridiaceae and genus Clostridium (Murray et
al. 2003). It is a motile gram positive sub terminal spore forming rod (Howerton et al. 2011,
Barbut et al. 2001, Koss et al. 2006) measuring 3-5 µm in length and 0.5µm in width
(Murray et al. 2003). It is a heterotrophic organism with an optimal growth temperature of
37°C in an anaerobic environment with peritrichous flagella (Murray et al. 2003). Over 400
strains of Clostridium difficile have been identified to date and only 20 toxic stains are known
to be seriously pathogenic towards humans or animals (Tonna et al. 2005, Hatheway 1990).
Colonies on culture media appear flat and slightly grey in colour with a ground glass
appearance. They have a distinctive ‘elephant house’ odour due to the production of iso-
valeric acid, iso-caproic acid and p-cresol, which are the products of various metabolic
pathways within the organism. They also produce catalase which can be used for differential
diagnosis of CD (Hatheway 1990, Tenover et al. 2011, Murray et al. 2003).
2.1 Pathogenesis
Clostridium difficile bacillus exists either as a vegetative cell or an endospore (Murray et al.
2003, Poutanen et al. 2004). The spores are highly resistant to physical and chemical
treatment with some cleaning agents known to enhance their resistance (Dancer 2009,

8
Howerton et al. 2011). CD is a nosocomial pathogen (hospital acquired) which is responsible
for Clostridium Difficile-associated diarrhoea (CDAD) and significant morbidity and
mortality amongst elderly people and patients in healthcare facilities (Roberts et al. 2008,
Tonna et al. 2005). It is also reported to be a cause of enteric diseases in animals like horses,
dogs, birds, pigs and rodents which are believed to act as reservoirs for CD (Kuijper et al.
2006). It is mainly acquired from the environment through the faecal-oral route and lives as a
commensal in the colon (Tonna et al. 2005, Cohen et al. 2010, Mulligan et al. 1979, Yakob et
al. 2013a). While most of the vegetative cells are killed by acid in the gut, spores, which are
resistant survive, establish themselves and colonise the gastrointestinal gut of people who
may be asymptomatic (Anonymous2004, Poutanen et al.2004)
The presence of up to 1012 organisms of normal flora in a gram of faeces composed of
predominantly Lactobacilli and enterococci help resist colonisation and stop multiplication of
C. Difficile in the colon (Tonna et al. 2005, Lopetuso et al. 2013). CDAD usually occurs
during or after antibiotic treatment (Bignardi 1998) by disrupting the normal gut flora
(dysbiosis), allowing CD from endogenous or exogenous origins to start multiplying and
proliferating (Barbut et al. 2001, Tonna et al. 2005, Lyerly et al.1998). Bile acids in the
stomach may also promote germination of the bacilli (Poutanen, Simor 2004).
Pathogenic strains of C. difficile produce two major glycosylating toxins; Toxin A
(enterotoxin) and Toxin B (cytotoxic) which are also its virulence factors and encoded on
pathogenicity locus 19.6 kb – PoLac(Deneve et al. 2009, Lyerly et al. 1998, Stabler et al.
2009, Voth et al. 2005). These toxins are encoded for by the genes tcdA and tcdB
respectively as seen in Figure 2 below. Both toxins are produced during the late lag and
stationary phases of growth which allows cells to become established within the host gut
before toxin production begins (Voth et al. 2005)

9
Figure 2: Showing the genetic arrangement of the C. difficile pathogenicity locus and proposed protein domain
structures of TcdA and TcdB (Voth et al. 2005).
The toxins A and B cause inflammation and damage to the mucosa and fluid secretions as its
characteristic pathology (Barbut et al. 2001, Poxton et al. 2001). They cause damage by
opening tight junctions between the cells of the intestine that result in increased vascular
permeability and haemorrhage. They also induce the production of tumour necrosis factor-
alpha (TNF-alpha) and pro inflammatory interleukins that cause a large inflammatory
response and ultimately the formation of pseudo membranes (Voth et al. 2005). The
pathogenesis of CD can be seen on fig. 3 below.

10
Figure 3: Pathogenicity of Clostridiumdifficile in the gut (Poutanen et al. 2004)
Other virulence factors also used by CD are the capsule (used as an antiphagocytic factor),
proteolytic enzymes (used to enhance mucus penetration), adhesins (involved in mucus and
cell adhesion)(Hennequin et al. 2001), fimbriae and flagella for penetration of mucus layer
(Deneve et al. 2009) . Lower levels of anti-toxin A IgG are associated with the severe form of
the disease (Tonna et al. 2005, Loo et al. 2011). People with a weakened immunity like HIV
infection may be prone to CD infection. Another factor is the emerging virulent strains of CD
as reported by (Cohen et al. 2010). The strain PCR ribotype 027 with genes encoding for
toxins A and B is an epidemic strain with an 18 base pair deletion in tcdC and is highly
virulent. It also has binary toxins called CDT (McDonald et al. 2005, Deneve et al. 2009)
which potentiates the toxicity of TcdA and TcdB leading to a more severe disease (Deneve et

11
al. 2009) . Within two weeks of colonisation with CD, cells of CD are shed in stool (Yakob et
al. 2013b).
2.2 Sources of Infection
Most studies have reported that people infected with CD shed up to 107 of CD per gram of
faeces into the environment. This is believed to be a source of CD infection (Best et al.
2010). The isolation of CD from skin sites of patients of CDAD even after the resolution of
diarrhoea is well documented (Rutala et al. 2013). These sites are the potential sites of
transmission between nurses, housekeepers and other patients (Rutala et al. 2013, Bobulsky
et al. 2008). Roberts et al. 2005 demonstrated that the environment is contaminated by the
use of nebulisers, the movement of people and bed making among others which liberate
aerosols into the environment as summarised in Figure 4 below (Roberts et al. 2006). This
means that these activities can lead to the contamination of air, food and fomites with CD if
present (Best et al. 2010, Al Saif et al. 1996). (Al Saif et al. 1996) noted the presence of CD
on vegetables - 2.4%, soil samples - 21%, river and lake water - 40 – 81.2%, hospital
environment – 20%, and nursing homes at 2.2%.

12
Figure 4: Overview of potential sources of Clostridiumdifﬁcile transmission. (Donskey 2010)
2.3 Infection Prevention
Infection control teams and other healthcare workers are faced with a challenge to control CD
infection. Prevention of CDI is delivered by preventing or stopping patient exposure to the
organism or ensuring that the patient’s gut flora is not disrupted and left susceptible to CDI
(HPA 2006). Several recommendations and guidelines have been rolled out over the years
which most healthcare providers including Airedale NHS trust have adopted (Gerding et al.
2008, Dancer 2009, Cohen et al. 2010, Stuart et al. 2011). Amongst the strategies
implemented are those aimed at targeting the environment, hospital personnel hand hygiene,
prevention of ingestion of spores and minimising antimicrobial exposure (good antimicrobial
stewardship) (Stuart et al. 2011, MacLeod-Glover et al. 2010). The use of C. difficile toxoid
vaccine is known to give high toxin A IgG in humans and confers immunity against diarrhoea
due to CD (Tonna, Welsby 2005, Kyne et al. 2001, Aboudola et al. 2003). It has been
extensively reported that CD spores survive the use of hand hygiene alcohol based gels and

13
cleaning detergents (Gerding et al. 2008). The spores also survive exposure to heat, acids
and most antibiotics (Rutala, Weber 2013). At Airedale NHS trust, the infection prevention
team monitor the severity of CDI, laboratory results, adherence to current antibiotic policy,
management of faecal contaminated laundry, hand hygiene and general and deep cleaning of
wards and other facilities (Charlesworth 2012a).
Isolation of suspected cases of CD infection helps to minimise the spread of the infection to
other wards and patients. This also helps the team to confine the infection (Charlesworth
2012a, Cohen et al. 2010).
The use of chlorine based cleaning agents is significant in reducing the contamination of
surfaces with CD (Wilcox et al. 2003). (Dancer 2009) reported traces of CD being found
after disinfection with bleach. These remnants of CD could be potential sources of infection
for patients being admitted afterwards (Wilcox et al. 2003, Rutala, Weber 2013) . Other
detergents in use in different hospitals are those whose active agents are acidified nitrite, par
acetyl ions, glutaraldehyde, alcohol, most of which are hazardous, and known to cause
asthma and dermatitis (Faise et al. 2010, MacLeod-Glover, Sadowski 2010). Therefore,
consideration of safety, effectiveness and cost needs to be made in choosing a suitable and
reputable detergent to use. For this reason, Airedale General Hospital changed from using a
hypochlorite based detergent to a chlorine dioxide one called Tristel. It is non-toxic, non-
flammable, and sporicidal (Faise et al. 2010).
3 MATERIALS AND METHODS
3.1 Air Sampling
Air samples were collected from wards with colonised or CDI patients before and/or after a
deep cleaning exercise and hospital corridors using a portable air sampler - AES Sampl’air

14
Lite from BioMérieux, Basingstoke, United Kingdom. Information on the CDI of ward
occupants at the time of sampling was obtained through infection prevention control team.
Following air sampler charging and head cleaning using 70% alcohol, the procedure from
Airedale NHS foundation Trust pathology services - Microbiology department’s protocol –
Appendix 1 was employed to prime a plate of commercially prepared Brazier’s Agar Medium
to ensure sterility (Crabtree 2012). A petri dish with Brazier’s media was placed on the clips
and the steel sampl’air head was placed on top. The sampl’air is run for 2 minutes.
The air sampler was then moved to different locations where samples were collected for 2
minutes (100 L/minute) at a velocity of 16.8 m/s. The samples were collected direct onto a
fresh commercially prepared Brazier’s Clostridium difficile Selective medium PB1055A,
Oxoid Limited, Basingstoke, United Kingdom. Plates were transported to the laboratory and
incubated anaerobically at 370C for 48 hours initially and read every 24 hours for the next 3
days if no growth was observed. The numbers of colonies grown on each plate were counted
and Appendix 2 was used to find N. This was used to calculate the level of airborne
contamination using the formula:
(N ÷ V)CFU /m3where N is equal to n (the number of colonies counted on a plate) used to
find N on appendix 1, V= volume derived by multiplying the sampling dilution by 0.1 m3
/minute.
3.2 Environmental Surface Swab Sampling
Two swabs from environmental sites were collected from door handles, floors, commodes,
bedrails, sinks, toilets, walls and bathrooms. One swab was transferred into a brain heart
infusion broth for the recovery of CD. They were vortexed for 1 minute and incubated
anaerobically for 48 hours at 370C. They were then subcultured onto Brazier’s CD selective
medium and incubated anaerobically at 370C for 48 hours and every 24 hours for the next 72

15
hours if no growth was observed. This was done for the pilot study only. The second swab
was inoculated direct onto Brazier’s media and processed as the first swab.
Sterile premoistened blue cellulose swabs (6) – (Polywipes) form Medical wire & equipment
Co. (Bath) Ltd, Wiltshire, United Kingdom as those used by (Best et al. 2010) were used for
collection of samples from surface areas of ward 6. For comparison, all the sites which were
sampled using a premoistened swab had a Polywipe swab collected as well. After collection,
the Polywipes were put in a sterile bag and transported to the laboratory where they were
placed in contact with the surface of Brazier’s media for one minute and the plate incubated
as described in section 3.2. A few free samples were used for this purpose.
3.3 Soil and Cow dung
Soil and cow dung samples were collected in a sterile screw capped bijou bottle. The sample
was alcohol shocked by adding an equal volume of absolute alcohol, vortexed and incubated
at room temperature for 30 – 60 minutes as done by (Best et al. 2010). The samples were then
centrifuged at 300 rpm for 5 minutes and two drops of the deposit were inoculated onto
Brazier’s media and incubated at 35 ±2 0 C in an anaerobic condition, for 48 hours initially
and read every 24 hours for the next 72 hours if no growth was observed.
3.4 Pilot Study
Comparing the use of brain heart infusion as an enrichment step with direct plating onto
Brazier’s media; the following procedure was used:
 Duplicate samples were collected from different sites in ward 2 which had a colonised
patient at the time using pre-moistened swabs (Physiological buffered saline)
 One swab was placed in brain heart infusion and processed following the proposed
procedure above for environmental surface swabs.

16
 The second swab was inoculated directly onto Brazier’s media and processed
following the proposed procedure above for environmental surface swabs.
3.5 Sampling and Processing Method Verification
(i) A Clostridium difficile positive sample was used for verification of the method
proposed in this study. It was used to test the ability of the two methods i.e. direct
plating and enrichment in brain heart infusion to recover C.D. from the spores on
the plate. CD was inoculated onto Brazier’s media, incubated at 35 ±2 0 C in an
anaerobic environment for 48hours. The plate was then placed on the open at
room temperature to allow it to dry and sporulate. After one week, two swabs
were collected from the plate and processed as per procedure for environmental
surface sampling above. During the process of inoculating the media, an air
sampler placed less than a metre below the bench was collecting air samples onto
Brazier’s media for 2 minutes. It was processed following the air sampling
procedure above.
(ii) This part of the study was conducted to show the ability of the method proposed
to recover C. difficile from surfaces at different concentrations. This was
performed following the procedure as done by (Buggy et al. 1983) with changes
as below.
A CD suspension of 0.5 Mcfarland standard equal to 1.5x108CFU/ml in normal
saline from a known Clostridium difficile positive sample was diluted by
transferring 1ml of the suspension into 9mls of normal saline and into the next
subsequent test tube to make a 1:10 dilution as shown in table 1 below.

17
Tube # 1 2 3 4 5 6 7
Conc.
of CD 1.5x108
CFU/ml 1.5x107
CFU/ml 1.5x106
CFU/ml 1.5x105
CFU/ml 1.5x104
CFU/ml 1.5x103
CFU/ml 1.5x102
CFU/ml
Smear
volume
100µl 100µl 100µl 100µl
Table 1: Concentrations of CD diluted 1:10 from test tube 1 to 7 and slide preparations for
method verification study.
A volume of 100µls from each suspension in the 1st, 3rd, 5th and 7th test tubes as
shown in table 1 were placed on glass slides and left at room temperature for a
week to dry and sporulate. Two pre-moistened swabs were used to collect a
sample from each glass slide and inoculated on to Brazier’s media. The swabs
were processed following the procedure above for processing environmental
surface swabs.
During the process of inoculating the media, an air sampler placed less than a metre below
the bench was collecting air samples onto Brazier’s media for 2 minutes. It was processed
following the air sampling procedure above. This was to isolate any aerosols (CD) liberated
during sample collection from surfaces where different concentrations of CD were placed.
3.6 Colony Identification of C. Difficile
Identification of CD was done by studying colony morphology, gram stain and biochemical
methods with the aid of a table 2 below.
C. difficile C. innocuum C. glycolicum C. sordelli/
bifementans
Odour + - - -
Lecithinase - - - +
UV fluorescence + + - -
API test
Table 2: differential tests for recognition of colonies of Clostridiumdifficile.

18
Colonies on Brazier’s medium appear yellowish to white, circular to irregular and flat with
rhizoid edge and a ground-glass appearance as described by (Beaugerie et al. 2003, Al Saif,
Brazier 1996). Screening colonies subcultured on Fastidious Anaerobe Agar (FAA) under
long-wave ultra violet light (365nm), shows yellow-green fluorescence. They produced
distinctive horse manure like odour (Murray et al. 2003). Gram stain showed gram positive to
variable thin rods 3-5µm by 0.5 µm. Colonies that fit this criteria were further tested by
API(Analytical Profile Index) test (a commercial system used to identify bacteria using
biochemical tests and a database) (Janda, Abbott 2002) called rapid ID 32 A V3.2 -
BioMérieux, Basingstoke. All isolates of CD positive samples by API test were also tested to
detect Clostridium difficile glutamate dehydrogenase antigen and toxins A and B in a single
reaction using C. Diff Quik Chek Complete – Alere limited, Cheshire, United Kingdom as
per manufacturer’s instructions. This was performed by modifying the method as performed
by (Friedman, Pollard et al. 2013) using a suspension of colonies instead of faeces. A sample
of the isolates grown on chocolate agar slopes was sent to Leeds general infirmary laboratory
for confirmation of C. difficile by PCR (Polymerase Chain Reaction) Ribotyping (Rupnik et
al. 2001) in keeping with the ANHST Microbiology protocol.
Analysis of the results was done using Microsoft Excel 2010.
4. RESULTS
(1) In the pilot study which was meant to evaluate the need for enrichment step with brain
heart infusion to be included or not, there was no CD isolated using the two methods – Direct
plating or enrichment with brain heart infusion. The next pilot study as in section 3.5 (i)
showed that brain heart infusion broth only isolated 50% of the know positive samples and
was thus its use in the study was stopped.

19
Clostridium difficile was recovered from all levels of concentrations using the proposed
method during method verification study with the lowest tested being 1.5x102 as shown in
table 3.
Tube # 1 2 3 4 5 6 7
Conc. of
CD 1.5x108
CFU/ml 1.5x107
CFU/ml 1.5x106
CFU/ml 1.5x105
CFU/ml 1.5x104
CFU/ml 1.5x103
CFU/ml 1.5x102
CFU/ml
Smear
volume
100µl
-
100µl
-
100µl
-
100µl
Recovery
of CD >100 colonies
-
80 colonies
-
10 colonies
-
3 colonies
API Test CD recovered - CD recovered - CD recovered - CD recovered
Table 3: Results of CD recovery for both direct plating and air sampling for method verification.
(2) The overall results of the study analysed using Excel 2010 are shown in figure 5 and 6
below.
Figure 5: a graph of Clostridiumdifficile isolates and the total number of samples collected from different
locations of Airedale NHS hospital and surrounding farms.
0
20
40
60
80
100
120
140
160
180
Totalnumberofsamples
Location
Number of Samples per location versus Clostridium difficile positives
Total# of samples
# of Positive samples

20
Out of 171 samples collected for the study, Clostridium difficile was isolated from 3 samples
(1.75%). Out of Nineteen air samples, one (5.26%) was positive for CD. The level of air
contamination was calculated as below:
Number of colonies (n) = 1; N (from appendix 1) = 1, total time = 2 minutes, volume=0.1;
Using (𝑁 ÷ 𝑉) ;(1 ÷ 2 × 0.1) CFU / m3 = 5 CFU / m3
The other 2 (2.35%) isolates out of 85 swabs were from ward 6 floor with a CD infected
patient.
During the comparative study of Polywipes against normal swabs (6), 1 colony of CD was
isolated using Polywipes from the floor. No CD was isolated from all the other surfaces by
Polywipes or normal swabs.
As seen from the data on figure 6 below, there was no CD isolated from cow manure and soil
samples.
Figure 6: showing different types of samples and the number of CD isolates.
19
85
39
28
171
1 2 0 0 3
0
20
40
60
80
100
120
140
160
180
200
Air samples Swab
samples
Soil samples Cow dung Total
Totalnumberofsamples
Sample type
Number of Samples testedversusClostridiumdifficile
positives
Total # of samples
# of Pos. samples

21
The three isolates were tested for Clostridium difficile glutamate dehydrogenase antigen and
toxins A and B and PCR Ribotyping was tested at Leeds general infirmary Hospital. Isolates
were assigned novel ribotypes (RT) according to Brazier’s nomenclature.
The results are shown in table 4 below.
Sample I.D. API test GDH Test
Antigen Toxin
PCR Ribotyping
069-08-01 CD Isolated + ± 002
111-01-01 CD Isolated + + 027
155-01-01 CD Isolated + - pending
Table 4: Results of C. Difficile isolates:
No C. difficile was seen on any of the control (QC) air samples from ward and corridors
(n=4).
5. DISCUSSION
The purpose of the study was to determine the prevalence of C. difficile in the hospital
environment in order to understand the extent of CD contamination which other patients may
be exposed to during hospital stay.
The hospital environment is a major source of infection where C. difficile spores can be found
for months, due to their resistance to heat and some disinfectants (Otter et al. 2011, Weber et
al. 2010, McFarland et al. 1989). While spores may remain in a dormant state for up to 40
days(Mulligan et al. 1979), providing a reservoir for new infections, vegetative forms of C
difﬁcile survives for up to 15 minutes on dry surfaces in room air, or may remain viable for
up to 6 hours on moist surfaces (Otter et al. 2011). Infected patients and asymptomatic
carriers may serve as reservoir for the organism which may act as an exogenous source of CD
to susceptible individuals (Roberts et al. 2008, Vaishnavi, Singh 2012). Medical devices
including portable bed commode and electronic rectal thermometers have been linked to the

22
transmission of CD in the hospital (Weber et al. 2010). Health workers’ hands have also been
reported to help in the transfer of contamination between patients or inanimate objects (Otter
et al. 2011, McFarland et al. 1989, Rutala, Weber 2013).
The prevalence of Clostridium difficile in a hospital environment is now well documented
with reported surface contamination rates ranging from 2% -59%(Kaatz et al. 1988, Kim et
al. 1981, McFarland et al. 1989, Al Saif, Brazier 1996, Martirosian 2006, Malamou-Ladas et
al. 1983, Titov et al. 2000, Vaishnavi, Singh 2012) air contamination from 0%-12%(Best et
al. 2010, Roberts et al. 2008, Kim et al. 1981), soil contamination of 21%-37%(Al Saif,
Brazier 1996, Simango 2006), and CD contamination in cow and its environment between
1%-3.4% (Al Saif, Brazier 1996, Simango 2006, Koene et al. 2012).
In this study, we established the overall prevalence rate of Airedale General Hospital
environment as 1.75% (3/171) with 5.26% (1/9) prevalence of CD in air samples, 2.35%
(2/85) of CD isolation from environmental surfaces and 0% from soil (=39) and Cow dung
(=28). The prevalence of CD in this study is consistent with previous studies but a few. The
difference between this study and a few of those studies with ridiculously high prevalence
rates lies in the different sampling and culturing methods used for the study and that sampling
in their case was done during a CDI outbreak. The rate of CD isolation increases when
samples are collected in areas where carriers have diarrhoea (outbreak) and is reduced in
those with non-known carriers (asymptomatic) (Kim et al. 1981, Faires et al. 2013).(Roberts
et al. 2008) used an enrichment media for air samples and had high rates of isolation of CD.
The use of enrichment broth with 1% sodium taurocholate increases the isolation of CD from
the samples as reflected in the results of their isolation rate (Martirosian 2006, McFarland et
al. 1989, Vaishnavi, Singh 2012, Howerton et al. 2011, Buggy et al. 1985). We did not use
enrichment broth with 1% sodium taurocholate due to budgetary constraints. We however
used enrichment broth (general) in our pilot study (section 3.4 and 3.5 i) and discontinued

23
due to results which indicated that it did not have an added advantage over direct plating. It
also resulted in overgrowth of other bacteria due to its luck of selective advantage (Buchanan
1984) which made it difficult to study a colony of interest. For these reasons, we only used
direct plating.
We isolated one toxigenic CD ribotype 027(reported using Brazier’s nomenclature)(Brazier
1998, Rousseau et al. 2011) from swab samples from ward 6 surfaces where the patient was
diagnosed with non-toxigenic CD ribotype 027 common to Airedale and the Yorkshire region
as shown in figure 7 below.
Figure 7: thedistributions of C. difficile ribotypes within Yorkshire and Humber region in England (April 2007-2011)
(Wilcox 2012).
We also isolated a non-toxigenic CD with results of ribotype yet to come back from leads
general hospital. The isolates were both from swabs collected from ward 6 floor in a space of
two weeks when the patient was still having diarrhoea. While the one isolates was of the
same ribotype (patient and environment), we could not conclude that they were linked to each

24
other due to the discordant toxigenic results. Since the patient was diagnosed with non-
toxigenic CD before coming into ward 6, toxigenic isolates from ward 6 floor may not be
from the patient. There is a possibility that the room may have been contaminated before and
not been decontaminated successfully. Some studies used multi-locus variable-number
tandem-repeat analysis (MLVA) to discriminate or link the isolates from the environment and
patients or animals (Best et al. 2010, Indra et al. 2008, van den Berg et al. 2007, Hota 2004).
Our isolates were not tested by MLVA because of the high cost of the test and that it was
beyond the scope of this study. However, the second isolate which was non-toxigenic may
have come be the same strain to that of the patient in which case it may indicate
environmental contamination from the symptomatic patient. In the absence of PCR ribotype
result, this conclusion cannot be made.
There was no CD isolated from any of the surfaces near other symptomatic patients except
the floor in ward 6 presumably due to good infection prevention practices. Compliance to
hand hygiene practices by health workers and visitors in patients’ rooms may have resulted in
less contamination transfer of CDI from patients to inanimate objects like door handles. It
may also indicate that the disinfectant (Tristel) being used on these surfaces is effective
against vegetative and spore forms of CD and used consistently as also reported by (Rutala,
Weber 2013). Even though the compliance with environmental cleaning was not measured,
the isolation of CD from the floor may indicate lack of better and more direct cleaning which
reduces the burden of microorganisms (Dancer 2009). Disinfectants do have a minimum
exposure time (Faise et al. 2010) and that these results may indicate that this was not met
especially in this case that CD was isolated twice from the floor of the same ward.
Our method verification study (section 3.5 ii) verified that the method was able to isolate CD
from surfaces with a lowest CD concentration of 1.5x102CFU/ml which was tested. However
we did not establish the lowest concentration that the method could isolate CD from. It is

25
possible that the method failed to isolate CD from the surfaces with lower contamination
levels than the minimum that we could have established. This could explain our lower
prevalence rate reported. We managed to obtain for free the highly recommended Polywipes
(Best et al. 2010) for sample collection which we used in parallel with swabs. We isolated 1/6
Polywipes and 0/6 from swabs. The Polywipes showed that they were more sensitive and that
they sampled a much wider surface which increased the chances of isolating CD. We could
not purchase any for purposes of this study due to excessive cost, but managed to use the free
ones as a small comparison to obtain and use them for this study due to budgetary constraints.
We isolated toxigenic CD ribotype 002 from air sample in the hospital corridors. This strain
is common to Airedale general hospital and the Yorkshire region as shown in figure 7
(Wilcox 2012). Figure 7 also shows that the strain was first reported in this region in 2008
and figures have stayed consistent. At a contamination level of 5 CFU / m3 isolated from the
corridor, it was the lowest detectable contamination level which resulted from 1 colony. In
our sample verification method study (section 3.5 ii) the lowest air contamination level of 15
CFU / m3 (table 3) was verified. However the method managed to isolate CD at a much
lower air contamination level (5 CFU / m3) showing that it was highly sensitive.
No CD was isolated from air samples from wards with CDI patients or outside the hospital.
Contrary to the findings of other studies (Best et al. 2010, Roberts et al. 2008) where they
reported isolating CD from air samples sampled close to CDI or symptomatic patients, we
isolated CD away from the CDI patients (corridors) and did not isolate it from air close to
CDI patients. Failure to identify CD from air samples close to symptomatic patient may
indicate that the air was not contaminated with CD.
Activities like bed making, movement of people, swinging doors open and close do help to
disperse the spores around (Best et al. 2010, Roberts et al. 2006). It is possible that the air at

26
sampling time had undetectable levels of CD contamination due to little activity by the bed
ridden immobile patients. We noted that an increase in sampling time for more than 2
minutes could have boosted the isolation rate.
We concluded that the isolation of CD from air samples in the hospital corridors indicated a
risk of CD contamination through air at a distance away from symptomatic patients even
though the level of air contamination at 5 CFU / m3 was low presumably due to good
ventilation.
The results also showed that soil and cow dung from part of the hospital environment
including the farm pose no potential risk as an exogenous source of CDI for the patients. Our
results are, in many ways, consistent with those of previous studies that have examined the
prevalence of CD in cow (Al Saif, Brazier 1996, Kim et al. 1981) who found no CD in them
and their environment. However, there is a possibility that vegetative bacilli of CD may have
been present in some of our fresh samples of Cow dung. The procedure of alcohol shock used
in processing them may have killed the CD as noted by (Chankhamhaengdecha et al. 2013)
that alcohol kills all bacteria including vegetative cells of CD except spores. This then may be
the reason reports of CD prevalence in cows may be reported lower. It may be necessary to
carry out a two arm study to test treated and untreated cow dung to confirm this hypothesis.
This study also demonstrated the inefﬁcacy of standard cleaning and disinfection procedures
against CD spores more so from the floor.
6. CONCLUSION
It is evident from the study results that inanimate objects in an environment of a person with
CDI can get contaminated by CD. Cleaning and decontamination of these areas with the right
detergents eradicate or reduce the contamination. The isolation of CD from air samples in the
hospital corridors also underlines the sporadic presence of CD in the air away from a CDI

27
patient. There was an indication that the cleaning detergents (Triste) in use at Airedale
hospital were effective and that there is need of intensifying the cleaning protocol in order to
be consistent and thorough. This conclusion was made due to the all-clear results from ward 2
where the CDI patient was admitted to before moving to ward 6 and that no CD was isolated
from all other sites except the floor in ward 6. More studies need to be carried out to include
the use of enrichment broth with 1% sodium taurocholate for better recovery of CD to help
report accurate prevalence of CD as recommended by a number of studies (Akhi et al. 2011,
Martirosian 2006). The use of Polywipes for sampling environmental surfaces would also be
recommended as it samples a lager surface area and is better than normal swabs (Best et al.
2010).
7. SOURCES OF MATERIALS AND MANUFACTURERS
1. C. Diff. Quik Check Complete, Alere limited, Stockport, Cheshire, SK7 5BW,
England.
2. Brazier’s medium, Fastidious Anaerobe Agar (FAA), Chocolate agar slopes,
Brain heart infusion, Oxoid Limited, Wade road, Basingstoke, England.
3. API rapid ID 32 A, AES Sampl’air Lite, Biomerieux UK ltd, Grafton Way,
Basingstoke, RG22 6HY, Hampshire, England.
4. Polywipes, Medical Wire & Equipment Co. Limited, Wiltshire, SN13 9RT,
England.
5. Swabs,
6. Gram stain kits, Pro-Lab diagnostics,unit 7 westwood court, Cheshire, CH64
3UJ.

28
8. ACKNOWLEDGEMENTS
This project was undertaken as part of the BSc honours degree project in Biomedical
Sciences, conducted at Airedale NHS General Hospital between July and September, 2013
and funded by the Trust with supplement funding from the University of Central
Lancashire. I thank God for the wisdom and perseverance that he has bestowed upon me
during this research project, and indeed, throughout my life: I can do anything through him
who gives me strength.
I would like to express my thanks to Suz Donald, who gave me the chance to work on this
research as part of my honours degree project.
I would also like to express sincere appreciation to my supervisor, Dr Laura McShane, for her
guidance throughout this project. Our discussions always helped me to focus my mind in
making some of the key decisions, Kathryn Moorhouse and Danielle North for their technical
support, guidance, and advice throughout the research project, as well as their pain-staking
effort in proof reading the drafts. I am indebted to most of biomedical scientists in the
department of Microbiology, for being there for me when my supervisors were on holiday.
James Stickland and the Infection prevention team for the information and coordination of
ward sampling. I thank Medical wire & equipment for providing us with free samples of
highly recommended Polywipes for use in this study. I also would like to thank farm mangers
of farms around Airedale general hospital and especially Barry for letting us into their farms
to collect samples used in this study.
Lastly, I offer my regards and blessings to all of those who supported me in any respect
during the completion of the project.

29
9. REFERENCES: -
1. "NationalClostridium difficileStandards Group: Report to the Department of Health", 2004,
Journal of Hospital Infection, vol. 56, Supplement 1, no. 0, pp. 1-38.
2. Aboudola, S., Kotloff, K., L., Kyne, L., Warny, M., Kelly, E., C., Sougioultzis, S.,
Giannasca, P.J., Monath, T., P. & and Kelly, C., P. 2003, "Clostridium difficile vaccine
and serum immunoglobulin G antibody responce to Toxin A", Infection and immunity,
vol. 71, no. 3, pp. 1608-1610.
3. Akhi, T., Mohammad, Pirzade, T. & Gojazade, M. 2011, "Antimicrobial susceptibility of
Clostridium difficile isolated from different sources of Imam Reza Hospital, Tabriz.",
African Journal of Microbiology Research, vol. 5, no. 19, pp. 2946-2949.
4. Al Saif, N., Brazier, J., S. 1996, "The distribution of Clostridium difficile in the
environment of South Wales", Journal of medical microbiology, vol. 45, no. 2, pp. 133-
137.
5. Barbut, F., Petit, J., C. 2001, "Epidemiology of Clostridium difficile-associated infections",
Clinical Microbiology and Infection, vol. 7, no. 8, pp. 405-410.
6. Bartlett, J.G. 2008, "Historical Perspectives on Studies of Clostridium difficile and C.
difficile Infection", Clinical Infectious Diseases, vol. 46, no. Supplement 1, pp. S4-S11.
7. Bartlett, J.G. 1994, "Clostridium difficile: History of Its Role as an Enteric Pathogen and
the Current State of Knowledge About the Organism", Clinical Infectious Diseases, vol.
18, no. Supplement 4, pp. S265-S272.
8. Beaugerie, L., Flahault, A., Barbut, F., Atlan, P., Lalande, V., Cousin, P., C adilhac, M. &
Petit, J., C. 2003, "Antibiotic-associated diarrhoea and Clostridium difficile in the
community", Alimentary Pharmacology & Therapeutics, vol. 17, no. 7, pp. 905-912.
9. Best, E., L., Fawley, W.,N., Parnell, P. & Wilcox, M., H. 2010, "The Potential for
Airborne Dispersal of Clostridium difficile from Symptomatic Patients", Clinical
Infectious Diseases, vol. 50, no. 11, pp. 1450-1457.
10. Bignardi, G., E. 1998, "Risk factors for Clostridium difficile infection", Journal of
Hospital Infection, vol. 40, no. 1, pp. 1-15.
11. Bobulsky, G., S., Al-Nassir, W., N., Riggs, M., M., Sethi, A., K. & Donskey, C., J. 2008,
"Clostridium difficile Skin Contamination in Patients with C. difficile–Associated
Disease", Clinical Infectious Diseases, vol. 46, no. 3, pp. 447-450.
12. Brazier, J., S. 1998, "The epidemiology and typing of Clostridium difficile", journal of
antimicrobial chemotherapy, vol. 41, pp. 47-57.
13. Buchanan, A., G. 1984, "Selective Enrichment Broth Culture for Detection of
Clostridium difficile and Associated Cytotoxin", Journal of clinical microbiology, vol.
20, no. 1, pp. 74-76.

30
14. Buggy, B., P., Wilson, K., H., Fekety, R. 1983, "Comparison of methods for recovery of
Clostridium difficile from an environmental surface.", Journal of clinical microbiology,
vol. 18, no. 2, pp. 348-352.
15. Buggy, B., P., Hawkins, C., C., Fekety, R. 1985, "Effect of adding Sodium Taurocholate
to selective media on recovery of Clostridium difficile from Environmental Surfaces.",
Journal of medical microbiology, vol. 21, no. 4, pp. 636-637.
16. Chankhamhaengdecha, S., Hadpanus, P., Aroonnual, A., Ngamwongsatit, P.,
Chotiprasitsakul, D., Chongtrakool, P., Janvilisri, T. 2013, "Evaluation of multiplex PCR
with enhanced spore germination for detection of Clostridium difficile from stool
samples of the hospitalised patients", BioMed research international, vol. 2013, no.
875437, pp. 1-6.
17. Charlesworth, A. 2012a, "Management of Clostridium difficile", Airedale NHS
Foundation Trust, Sharepoint, vol. 1, no. 2.1, pp. 1-14.
18. Charlesworth, A. 2012b, Update of actions to reduce the incidence of Clostridium
difficile at Airedale NHS foundation trust, Airedale NHS foundation trust, Airedale NHS
General Hospital.
19. Cohen, S., H., Gerding, D., N., Johnston, S., Kelly, C., P., Loo, V., G., McDolnald, C., L.,
Pepin, J., Wilcox, M., H. 2010, "Clinical practice guidlines for clostridium difficile
infection in adult: 2010 update by the society for healthcare epidemiology of america
(SHEA) and the infectious diseases society of America (IDSA)", Infection Control and
Hospital Epidermiology, vol. 31, no. 5, pp. 431-455.
20. Crabtree, A. 2012, "Micro_LPT_68 Use of AES Portable air sampler", Airedale
Foundation Trust, Pathology Services, Department of Microbilogy, , no. 1.2, pp. 1-9.
21. Dancer, S., J. 2009, "The role of environmental cleaning in the control of hospital-
acquired infection", Journal of Hospital Infection, vol. 73, pp. 378-385.
22. Deneve, C., Janoir, C., Poilene, I., Fantinato, C., Collignon, A. 2009, "New trends in
Clostridium difficile virulence and pathogenensis", International Journal of
antimicrobial agents, vol. 33, no. 51, pp. S24 S28.
23. Donskey, C., J. 2010, "Preventing transmission of clostridium difficile: is the answer
blowing in the wind?", Clinical Infectious Diseases, vol. 50, no. 11, pp. 1458-1461.
24. Faires, M., Pearl, D., Berke, O., Reid-Smith, R., Weese, J. 2013, "The identification and
epidemiology of methicillin-resistant Staphylococcus Aureus and Clostridium difficile in
patient rooms and the ward environment", BMC Infectious Diseases, vol. 13, no. 1, pp.
342.
25. Faise, A., P., Bradley, C., R., Wilkinson, M., A. 2010, Assesment of the activity of Tristel
Fuse against Clostridium Difficile., Research edn, Hospital Infection Research
Laboratory, Birmingham.

31
26. Friedman, N., Pollard, J., Stupart, D., Knight, D., Khajehnoori, M., Davey, E., Parry, L.,
Riley, T. 2013, "Prevalence of Clostridium difficile colonization among healthcare
workers", BMC Infectious Diseases, vol. 13, no. 1, pp. 459.
27. George, L., W., Rolfe, R., D., Mulligan, E., Maury, Finegold, S., M. 1979, "Infectious
Diseases 1979: Antimicrobial Agent-Induced Colitis: An Update", The Journal of
Infectious Diseases, vol. 140, no. 2, pp. 266-268.
28. Gerding, D., N., Muto, C., A., Owens, R., C. 2008, "Measures to Control and Prevent
Clostridium difficile Infection", Clinical Infectious Diseases, vol. 46, no. Supplement 1,
pp. S43-S49.
29. Hatheway, C., L. 1990, "Toxigenic Clostridia", Clinical microbiology reviews, vol. 3, pp.
66-98.
30. Hennequin, C., Porcheray, F., Waligora-Dupriet, A., Collignon, A., Barc, M., Bourlioux,
P., Karjalainen, T. 2001, "GroEL (Hsp60) of Clostridium difficile is involved in cell
adherence", Microbiology, vol. 147, no. 1, pp. 87-96.
31. Hill, K.A., Collins, J., Wilson, L., Perry, J.D., Gould, F.K. 2013, "Comparison of two
selective media for the recovery of clostridium difficile from environmental surfaces",
Journal of hospital infection, vol. 83, pp. 164-166.
32. Hota, B. 2004, "Contamination, disinfection, and cross-colonization: are hospital surfaces
reservoirs for nosocomial infection?", Clin Infect Dis, vol. 39, no. 8, pp. 1182-1189.
33. Howerton, A., Ramirez, N., Abel-Santos, E. 2011, "Mapping Interactions between
Germinants and Clostridium difficile Spores", Journal of Bacteriology, vol. 193, no. 1,
pp. 274-282.
34. HPA 2012, Voluntary surveillance of clostridium difficile in England Wales and Northern
Ireland 2012, Health protection agency, London.
35. HPA 2006, Clostridium difficile: Findings and recommendations from a review of the
epidemiology and a survey of Directors of Infection Prevention and Control in England,
Health Protection Agency.
36. Indra, A., Huhulescu, S., Kuijper, E., J. 2008, "characterisation of clostridium difficile
isolates using capillary gel electrolysis-based PCR ribotypeing", Journal of medical
microbiology, vol. 57, no. 11, pp. 1377-1382.
37. Janda, J., Michael, Abbott, S., L. 2002, "Bacterial Identification for Publication: When Is
Enough Enough?", Journal of clinical microbiology, vol. 40, no. 6, pp. 1887-1891.
38. Kaatz, G.W., Gitlin, S.,D., Schaberg, D., R., Wilson, K., H., Kauffman, C., A., Seo, S.,
M., Fekety, R. 1988, "Acquisition of Clostridium difficile from the hospital
environment", Am J Epidemiol, vol. 127, no. 6, pp. 1289-1294.
39. Kim, K., H., Fekety, R., Batts, D., H., Brown, D., Cudmore, M., Silva, J., Waters, D.
1981, "Isolation of Clostridium difficile from the Environment and Contacts of Patients

32
with Antibiotic-Associated Colitis", Journal of Infectious Diseases, vol. 143, no. 1, pp.
42-50.
40. Koene, M., G., Mevius, D., Wagenaar, J., A., Harmanus, C., Hensgens, M., P.,
Meetsman, A., M., Putirulan, F., F., Kuijper, E., J. 2012, "Clostridium difficile in Dutch
animals: their presence, characters and similaries with human animals", Clinical
Microbiology and Infection, vol. 18, no. 8, pp. 778-784.
41. Koss, K., Clark, M., A., Sanders, D., S., A., Morton, D., Keighley, M., R., B., Goh, J.,
2006, "The outcome of surgery in fulminant Clostridium difficile colitis", Colorectal
Disease, vol. 8, no. 2, pp. 149-154.
42. Kuijper, E., J., Coignard, B., Tüll, P. 2006, "Emergence of Clostridium difficile-
associated disease in North America and Europe", Clinical Microbiology and Infection,
vol. 12, pp. 2-18.
43. Kyne, L., Warny, M., Qamar, A., Kelly, C.P. 2001, "Association between antibody
response to toxin A and protection against recurrent Clostridium difficile diarrhoea", The
Lancet, vol. 357, no. 9251, pp. 189-193.
44. Loo, V., G., Bourgault, A., Poirier, L., Lamothe, F., Michaud, S., Turgeon, N., Toye, B.,
Beaudoin, A., Frost, E., H., Gilca, R., Brassard, P., Dendukuri, N., Béliveau, C.,
Oughton, M., Brukner, I., Dascal, A. 2011, "Host and Pathogen Factors for Clostridium
difficile Infection and Colonization", N Engl J Med, vol. 365, no. 18, pp. 1693-1703.
45. Lopetuso, L., Scaldaferri, F., Petito, V., Gasbarrini, A. 2013, "Commensal Clostridia:
leading players in the maintenance of gut homeostasis", Gut Pathogens, vol. 5, no. 1, pp.
23.
46. Lyerly, D., M., Krivan, H., C., Wilkins, T., D. 1998, "Clostridium difficile:its disease and
toxins.", Clinical microbiology reviews, vol. 1, no. 1, pp. 1-18.
47. MacLeod-Glover, N., Sadowski, C. 2010, "Efficacy of cleaning products for C difficile:
Environmental strategies to reduce the spread of Clostridium difficile–associated
diarrhea in geriatric rehabilitation", Canadian Family Physician, vol. 56, no. 5, pp. 417-
423.
48. Malamou-Ladas, H., O'Farrell, S., Nash, J.Q., Tabaqchali, S. 1983, "Isolation of
Clostridium difficile from patients and the environment of hospital wards", J Clin
Pathol, vol. 36, no. 1, pp. 88-92.
49. Martirosian, G. March 2006, "Recovery of Clostridium difficile from Hospital
Environments", Journal of clinical microbiology, vol. 44, no. 3, pp. 1202-1203.
50. Martirosian, G. 2006, "Recovery of Clostridium difficile from Hospital Environments",
Journal of clinical microbiology, vol. 44, no. 3, pp. 1202-1203.
51. Mayfield, J., L., Leet, T., Miller, J., Mundy, L., M. 2000, "Environmental Control to
Reduce Transmission of Clostridium difficile", Clinical Infectious Diseases, vol. 31, no.
4, pp. 995-1000.

33
52. McDonald, C., L., Coignard, B., Dubberke, E., Song, X., Horan, T., Kutty, P., K. 2007,
"Recommendations for Surveillance of Clostridium difﬁcile–Associated Disease",
Infection Control and Hospital Epidermiology, vol. 28, no. 2, pp. 140-145.
53. McDonald, L., C., Killgore, G., E., Thompson, A., Owens, R., C., Kazakova, S., V.,
Sambol, S., P., Johnson, S., Gerding, D., N. 2005, "An Epidemic, Toxin Gene–Variant
Strain of Clostridium difficile", N Engl J Med, vol. 353, no. 23, pp. 2433-2441.
54. McFarland, L., V., Mulligan, M., E., Kwok, Richard, Y., Y., Stamm, W., E. 1989,
"Nosocomial Acquisition of Clostridium difficile Infection", N Engl J Med, vol. 320, no.
4, pp. 204-210.
55. Mulligan, E., Maury, Rolfe, D., Rial, Finegold, M., Sydney, George, L., W. 1979,
"Contamination of a hospital environment by Clostridium Difficile", Current
Microbiology, vol. 3, no. 3, pp. 173-175.
56. Murray, P., R., Baron, E., Jo, Jorgensen, J., H., Pfeller, M., A., Yolken, R., H. 2003,
Manual of clinical microbiology, Voolume 1, 8th edn, American Association for
Microbiology press, Washington.
57. Muto, C., A., Blank, M., Kathleen, Marsh, J., W., Vergis, E., N., O'Leary, M., M., Shutt,
K., A., Pasculle, A., W., Pokrywka, M., Garcia, J., G., Posey, K., Roberts, T., L.,
Potoski, B., A., Blank, G., E., Simmons, R., L., Veldkamp, P., Harrison, L., H., Paterson,
D., L. 2007, "Control of an Outbreak of Infection with the Hyper virulent Clostridium
difficile BI Strain in a University Hospital Using a Comprehensive "Bundle" Approach",
Clinical Infectious Diseases, vol. 45, no. 10, pp. 1266-1273.
58. Otter, J., A., Yezil, S., French, G., L. 2011, "The role played by contaminated surfaces in
the transmission of Nosocomial pathogens", Infection Control and Hospital
Epidemiology, vol. 32, no. 7, pp. 687-699.
59. Poutanen, S., M. & Simor, A., E. 2004, "Clostridium difficile-associated diarrhea in
adults", Canadian Medical Association journal, vol. 171, no. 1, pp. 51-58.
60. Poxton, I., R., McCoubrey, J. & Blair, G. 2001, "The pathogenicity of Clostridium
difficile", Clinical Microbiology and Infection, vol. 7, no. 8, pp. 421-427.
61. Roberts, K., Smith, C., Snelling, A., Kerr, K., Banfield, K., Sleigh, P., A. & Beggs, C.
2008, "Aerial Dissemination of Clostridium difficile spores", BMC Infectious Diseases,
vol. 8, no. 1, pp. 7.
62. Roberts, K., Hathway, A., Fletcher, L.A., Beggs, C.B., Elliott, M.W. & Sleigh, P.A. 2006,
"Bioaerosol production on a respiratory ward", Indoor and Built Environment, vol. 15,
pp. 35-40.
63. Rousseau, C., Lemée, L., Le Monnier, A., Poilane, I., Pons, J. & Collignon, A. 2011,
Prevalence and diversity of Clostridium difficile strains in infants.

34
64. Rupnik, M., Brazier, J., S., Duerden, B.I., Grabnar, M. & Stubbs, Simon, L., J. 2001,
"Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and
description of novel toxinotypes", Microbiology, vol. 147, no. 2, pp. 439-447.
65. Rutala, W., A & Weber, D., J. 2013, "Role of the hospital environment in disease
transmission, with a focus on Clostridium difficile", Healthcare Infection .
66. Simango, C. 2006, "Prevalence of Clostridium difficile in the environment in a rural
community in Zimbabwe", Transactions of the Royal Society of Tropical Medicine and
Hygiene, vol. 100, no. 12, pp. 1146-1150.
67. Stabler, R., A., He, M., Dawson, L., Martin, M., Esmeralda, V., Corton, C., Lawley, T.,
D., Sebaihia, M., Quail, M., A., Rose, G., Gerding, D., N., Gibert, M., Popoff, M., R.,
Parkhill, J., Dougan, G. & Wren, B., W. 2009, "Comparative genome and phenotypic
analysis of Clostridium difficile 027 strains provides insight into the evolution of a
hypervirulent bacterium.", Genome Biology, vol. 10, no. 9, pp. r102.
68. Stuart, R., L., Marshall, C., McLaws, M., Boardman, C., Russo, P., L., Harrington, G. &
Ferguson, J., K. 2011, "ASID/AICA position statement - infection control guidelines for
patients with Clostridium difficile infection in healthcare settings.", Healthcare Infection,
vol. 16, pp. 33-39.
69. Tenover, F., C., J o Baron, E., Peterson, L., R. & Persing, D., H. 2011, "Review
Laboratory Diagnosis of Clostridium difficile Infection Can Molecular Amplification
Methods Move Us Out of Uncertainty?", The journal of molecular Diagnostics, vol. 13,
no. 6, pp. 573-582.
70. Titov, L., Lebedkova, N., Shabanov, A., Tang, Y.J., Cohen, S.H. & Silva, J. 2000,
"Isolation and Molecular Characterization of Clostridium difficile Strains from Patients
and the Hospital Environment in Belarus", Journal of clinical microbiology, vol. 38, no.
3, pp. 1200-1202.
71. Tonna, I. & Welsby, P., D. 2005, "Pathogenesis and treatment of Clostridium difficile
infection", Postgraduate medical journal, vol. 81, no. 956, pp. 367-369.
72. Vaishnavi, C. & Singh, M. 2012, "Preliminary investigation of environmental prevalence
of Clostridium difficile affecting inpatients in a north Indian hospital.", Indian Journal of
Medical Microbiology, vol. 30, no. 1, pp. 89-92.
73. van den Berg, Renate, J., Schaap, I. & Kuijper, E., J. 2007, "Typing and subtyping of
Clostridium difficile isolates by using multple-locus variable-number tandem-repeat
analysis", Journal of medical microbiology, vol. 45, no. 3, pp. 1024-1028.
74. Voth, D., E. & Ballard, J., D. 2005, "Clostridium difficileToxins: Mechanism of Action
and Role in Disease", Clinical microbiology reviews, vol. 18, no. 2, pp. 247-263.
75. Weber, D., J., Rutala, W., A., Miller, M., Huslage, K. & Sickbert-bennett, E. 2010, "Role
of hospital surfaces in the transmission of emerging health care-associated pathogens:
Norovirus,Clostridium difficile, and Acinetobater species", Am J Infect Control, vol. 38,
pp. 25-33.

35
76. Wilcox, M. 2012, Clostridium difficile Ribotyping Network (CDRN) for England and
Northern Ireland annual report, Health protection Agency, London.
77. Wilcox, M., H., Fawley, W., N., Wigglesworth, N., Parnell, P., Verity, P. & Freeman, J.
2003, "Comparison of the effect of detergent versus hypochlorite cleaning on
environmental contamination and incidence of Clostridium difficile infection", Journal
of Hospital Infection, vol. 54, no. 2, pp. 109-114.
78. Yakob, L., Riley, T., Paterson, D. & Clements, A. 2013a, "Clostridium difficile exposure
as an insidious source of infection in healthcare settings: an epidemiological model",
BMC Infectious Diseases, vol. 13, no. 1, pp. 376.
79. Yakob, L., Riley, T., Paterson, D. & Clements, A. 2013b, "Clostridium difficile exposure
as an insidious source of infection in healthcare settings: an epidemiological model",
BMC Infectious Diseases, vol. 13, no. 1, pp. 376.
10. APPENDICES
Appendix1: Table for calculating the air contamination from the number of colonies isolated
from air samples.

36

THE PREVALENCE OF CLOSTRIDIUM DIFFCILE AT AIREDALE NHST ENVIRONMENT

Recommended

Recommended

More Related Content

What's hot

What's hot (19)

Similar to THE PREVALENCE OF CLOSTRIDIUM DIFFCILE AT AIREDALE NHST ENVIRONMENT

Similar to THE PREVALENCE OF CLOSTRIDIUM DIFFCILE AT AIREDALE NHST ENVIRONMENT (20)

THE PREVALENCE OF CLOSTRIDIUM DIFFCILE AT AIREDALE NHST ENVIRONMENT