Infection Control & Hospital Epidemiology
Methods Strains and growth conditions
Type strain Clostridium difficile NCTC 11209 was obtained from the National Collection of Type Cultures. The clinical strain ribotype 001/072 was isolated during the investigation. Cooked meat broth (Fluka, Sigma-Aldrich, St Louis, MO) was used to produce spore suspensions, and supplemented brain heart infu- sion (BHI) agar (Oxoid, Ontario, Canada) was used to germinate and quantify viable spores (BHISs). For growth of vegetative cells, the BHI agar was supplemented with 0.1% L-cysteine (Sigma) and 5% horse blood (TCS Biosciences, Buckingham, UK). For spore germination, 0.1% taurocholic acid sodium salt hydrate (Sigma) was added to the growth agar (BHIS/T). Anaerobic culturing conditions were used.
Spore production
A single colony of C. difficile was inoculated into each of 20 aliquots (10mL) of pre-reduced cooked meat broth, then incu- bated for 10 days at 37°C. After the incubation, cultures were combined and centrifuged at 3,300×g for 20 minutes. The spore pellet was resuspended in 5mL 95% ethanol for 20 minutes to inactivate any vegetative cells. The suspension was then cen- trifuged and washed in maximum recovery diluent (MRD) 3 times. The final pellet was resuspended in MRD and stored at 4°C until use. Spores were enumerated by spread plating onto BHIS and incubated anaerobically at 37°C for 48 hours.
Soiling effect on C. difficile spore recovery from NHS (100% cotton) sheet swatches
Bovine serum albumin (BSA) was used to simulate soiling in ‘clean’ (0.3g/L) and ‘dirty’ (3g/L) conditions according to BS EN 13704.13 Sterile NHS (100% cotton) sheet swatches (25cm2) were each inoculated with a 0.1-mL aliquot of 6 log10 colony-forming units per milliliter (cfu/mL) spore-test suspension, with a final concentration of either 30 g/L BSA or 3 g/L BSA, and air dried for 18 hours. The swatches were transferred to either a stomacher bag or to a 50-mL Falcon tube containing 10mL MRD, then were stomached in a paddle blender (LM40, Seward, UK) on high for 1 minute or were vortexed (ThermoFisher Scientific, Loughbor- ough, UK) on high at 40Hz for 5 bursts of 5 seconds. Then, 0.1mL of the supernatant was plated onto BHIS/T agar. All agar plates were incubated anaerobically at 37°C for 48 hours.
Simulated industrial laundering of C. difficile spores on 100% cotton sheets
Sterile swatches (25cm2) were each inoculated with 0.1mL C. difficile NCTC 11209 spore suspension containing 8 log10 cfu/mL spores. The swatches were air dried overnight for 18 hours. Before washing, the swatches were attached by a sterile safety pin to a single NHS (100% cotton) sheet and placed in a washer extractor (Schulthess 6166, Wolfhausen, Switzerland). Each load contained 4 inoculated swatches and 4 sterile swatches. The full load (6.5 kg) included 10 additional sterile NHS (100% cotton) sheets. The temperature in the drum was verified every 5 seconds by a data logger (Measurement Systems, Newbury, UK). The industrial detergent system (Spectrum EU, Washing Systems International, Cheshire, UK) is outlined in Table 1. A control wash was also conducted without the detergent, and the investigation was repeated with the clinical strain ribotype 001/072.
1407 After the wash cycle, each swatch was vortexed in 30mL MRD,
5 times for 5 seconds, then left in the MRD for 5 minutes. The enumeration of surviving spores was conducted by vacuum fil- tering (black 0.45 µm Whatman filter, GE Healthcare Life Sci- ences, Marlborough, MA). For detergent cycles, the entire 30mL suspension was filtered, and for control cycles, duplicate 0.1mL samples were filtered, both followed by duplicate 0.1mL samples from a 1 log10 serial dilution. Method validations showed that filtration was successful at neutralizing the detergent (data not shown). The filter membranes were transferred to pre-reduced BHIS agar plates and incubated anaerobically at 37°C for 48 hours.
Scanning electron microscopy (SEM) of spores on cotton after laundering
Cotton swatches were fixed and prepared for viewing by SEM using 2% glutaraldehyde (G7526, Sigma) in phosphate buffer. Individual swatches were mounted on 2.5-cm aluminum stubs and sputter coated with gold (S150B sputter coater, Edwards, Burgess Hill, UK). The stubs were viewed using a SEM (Evo HD 15, Carl Zeiss, Oberkochen, Germany) under high vacuum with a beam accelerating voltage of 5–7 kV for magnifications between 2,500 and 50,000× .
Ethical approval
Ethical approval for the study was obtained from (1) De Montfort University Health and Life Sciences’ Research Ethics Committee (reference: 714) and (2) ‘Nottingham 2’ NHS Research Ethics Committee (reference: 11/EM/0002). Local research and devel- opment permission was obtained from the trust responsible for the NHS hospital research site (reference: UHL10003). The par- ticipating hospital allowed access to a C. difficile isolation ward.
Media
Braziers CCEY selective agar (Lab160, LabM, Heywood, UK) was used to ensure that only C. difficile spores were isolated from the samples. The agar contained the selective antibiotic cycloserine and cefotoxin supplement (X093, LabM) and egg yolk emulsion (X073, LabM).
Storage and collection of the naturally contaminated sheets
To ensure a realistic quantification of C. difficile spore burden before washing, storage of sheets after use in the ward was the same as for standard infected linen. A set of experimental sheets were delivered weekly to the ward. The sheets were used on CDI patient beds and changed daily or when soiling had occurred. After use, the sheets were placed in alginate bags and sealed. The alginate bags were put into standard red ‘infected-linen’–labelled plastic, nonpermeable bags and stored in the ward’s used linen room. The bags were collected every 24 hours, in accordance with standard practice at the site, were transferred to an external waste compound overnight, and were collected the following morning for sampling.
Sampling
Soiled sheets were numbered, and regions soiled with fecal matter were identified. From these, three 25cm2 swatches were removed leaving adjacent 25cm2 soiled regions for sampling after washing. The sheet was placed back into the alginate bag, resealed with the
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