Infection Control & Hospital Epidemiology
1409
Fig. 3. Scanning electron microscopy (SEM) images of C. difficile spores on a NHS (100% cotton) sheets: swatch inoculated with 0.1mL 8 log10 cfu/mL test suspensions containing 3 g/L BSA. The swatch air dried for 24 hours and was then washed in a simulated healthcare wash at 71°C with industrial detergent (held at 71°C for 5 minutes). (A) spore, (B) many spores left on the swatch after washing, (C) further magnification area in (B), and (D) large clump of spores (soiling or coaggregation).
Whitney U test or a Kruskal-Wallis test was performed. Each investigation was repeated in triplicate on 2 separate occasions (n=6) unless otherwise stated.
Results
Significantly more spores were recovered from cotton swatches by the vortexing method than by the stomaching method, at both light and heavy levels of soiling (P≤.05) (Fig. 1). For the vor- texing method, 4.48 and 4.41 log10 cfu/25cm2 spores were recovered from heavily and lightly soiled swatches, respectively (original inoculum, 5 log10 cfu/25cm2). In the simulated laundry control cycle (without detergent), significantly fewer spores were recovered, compared to the ori- ginal inoculum (7 log10 cfu/25cm2), for both C. difficile NCTC 11209 (4.95 log10 cfu/25cm2; P≤.05) and ribotype 001/072 (5.27 log10 cfu/25cm2; P≤.05) (Fig. 2). In addition, the numbers of recovered spores after this laundry control cycle were not sig- nificantly (P > .05) different between the 2 strains. Without detergent, the effects of temperature and agitation alone did not reduce the number of spores by >2 log10 cfu/25cm2, leaving the swatches heavily contaminated with 4.95 log10 cfu/25cm2 C. difficile spores (Fig. 2). Regarding the level of cross contamination during the control
cycle, similar numbers of spores were recovered from previously sterile swatches for both NCTC 11209 and ribotype 001/072 (2.72 vs 2.89 log10 cfu/25cm2). In wash cycles that included detergent, recovery of spores for both strains was significantly (P≤.05) reduced (to 0–4 cfu/25cm2 for NCTC 11209 and 0–9 cfu/25cm2 for ribotype 001/072), with cross contamination to previously sterile swatches ranging from 0 to 8 cfu/25cm2 and from 0 to 14 cfu/25cm2 (Fig. 2).
Table 2. C. difficile Spores Recovered From Swatches (25cm2) of Naturally Contaminated (100% cotton) Sheets, Before and After Washing to Meet NHS Healthcare Policy Minimum Standards HTM 01-04 at Industrial Launderers (n=3)
Sample Mean Spore Count Range Standard Error Ribotype Prewash 51 cfu/25cm2
Postwash 33 cfu/25cm2 100 cfu/75cm2
2–158 …*
33 …*
001/072 001/072
Note. cfu, colony-forming units. aThe cfu per 75cm2 wasrecorded at maximum count for each sheet (≥100) where the three 30-mL recovered suspensions per sheet were filtered together.
an average spore load of 51 cfu/25cm2 (1.7 log10 cfu/25cm2), with a range of 2–158 cfu/25cm2 (Table 2). The post-wash swatches (washed at ≥71°C for 3 minutes plus 8 minutes mixing, dried, and finished) still had an average spore load of 33 cfu/25cm2 (1.25 log10 cfu/25cm2). The post-wash average spore load was calculated from a combined 90-mL sample for each sheet: >100 cfu/75cm2. Thus, the post-wash reduction was 18 cfu/25cm2 (or 0.45 log10 cfu/25cm2), only a 40% reduction in spore load after washing. Subsequent ribotyping of the isolates recovered before and after washing showed that they were indistinguishable and had only a single minor difference from ribotype 001/072 (PHE, Newcastle). This finding suggests that the spores recovered after
The presence of C. difficile spores on cotton swatches after laundering was confirmed by SEM (Fig. 3). The SEM images show clumps of C. difficile spores, potentially within the soiling with which they were inoculated (Fig. 3d). Possible spore lysis was also evident (Fig. 3c), possibly through detergent or thermal disin- fection or a combination of both. Swatches removed from the naturally contaminated sheets had
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