Successful Removal of Clostridioides Difficile Spores and Pathogenic Bacteria From a Launderable Barrier Using a Commercial Laundry Process

Abstract

Background:

To properly clean and disinfect hospital mattresses, bed manufacturers recommend a 3- to 6-step process to remove all pathogenic bacteria. An alternative is to use a removable barrier on the mattress, which is laundered after each use. The current study was to determine efficacy of a commercial laundry process in eliminating Clostridioides difficile (C diff) spores, Mycobacterium terrae (M terrae), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P aeruginosa), Klebsiella pneumoniae (K pneumoniae), and Escherichia coli (E coli) from a barrier.

Methods:

A test barrier received 3 unique microbial suspensions in separate locations, each suspension having a known quantity of specific microorganisms: C diff spores, M terrae, and a mixed suspension of MRSA, S aureus, P aeruginosa, K pneumoniae, and E coli. A wash load contained the test barrier and 11 additional ballast barriers. Various soils were spread onto the barriers to simulate heavy soiling that may occur in a wash load: Each barrier received a small amount of mixed soil, 50% received urine, 25% received blood, and 25% received a large amount of additional mixed soil. The load was laundered using 71°C (160°F) water, detergent, and chlorine bleach, with final drying at 71°C (160°F). After laundering, remaining colony-forming units (CFUs) of each microorganism were counted at the applied locations. Each test was replicated 3 times. Industry-accepted methods were used to produce suspensions, apply inoculum, and recover organisms after laundering.

Results:

Before laundering, test barriers contained at least 7.0 log₁₀ cfu/mL of each microorganism distributed over 103 cm². After laundering, in all cases, no residual CFUs were detected over the test area, resulting in greater than 6.0 log₁₀ reductions for every organism. (P < .05).

Conclusions:

Under extreme test conditions including the presence of soil, the laundry process removed all detectable pathogenic bacteria and spores from the barrier.

Introduction

Health care–acquired infections (HAIs) continue to be a problem in the United States and worldwide. The most recent data indicate some improvements in rates of HAIs; however, in the United States, there still were 687 000 HAIs in 2015, which resulted in 72 000 deaths.¹ While there continues to be a lot of efforts to improve hand hygiene, there is inadequate attention being given to disinfection of the hospital environment.² Numerous studies have linked the hospital mattress to HAIs including OXA-48-producing Klebsiella pneumoniae and CTX-M-15-producing Enterobacter cloacae.^3-5

Most mattress covers today are made of polyurethane coated fabric and are typically cleaned and disinfected by manually wiping the surface 1 time with a health care disinfectant. Currently used disinfectants include quaternary ammonia compounds, sodium hypochlorite (bleach), hydrogen peroxide, and peroxyacetic acid (peracetic acid), hydrogen peroxide/peroxyacetic acid combination, and phenolics. While the disinfectants kill bacteria in the laboratory on hard surfaces, their performance in actual use on soft, hygroscopic mattress covers is limited by variability in the manual process, insufficient soil removal, and difficulty maintaining specified disinfectant dwell time. The use of quaternary ammonia achieved only a 1 log₁₀ cfu reduction in bacteria.^6,7 Peroxyacetic acid use was associated with a 2 log₁₀ cfu reduction in pathogenic bacteria, but only a 1 log₁₀ cfu reduction in Clostridioides difficile (C diff).^8,9 Sodium hypochlorite use also failed to eliminate C diff from hospital surfaces (1 log₁₀ cfu reduction).^10,11

Only a small percentage of hospitals preclean the mattress and rinse off the disinfectants, as recommended by many mattress manufacturers.¹² The failure to rinse these disinfectants off the mattress may be the reason for increasing rates of mattress damage. One study of acute care hospitals in Canada showed 32.5% of mattresses were damaged, and another in the United States found 26% were damaged.^13,14 In 2017, the United States Food and Drug Administration (FDA), after receiving more than 700 reports of mattress failure, issued a safety notice advising hospitals to routinely inspect mattresses for damage.¹⁵ ECRI Institute described mattress contamination and damage as 1 of their top health hazards for 2018 and 2019.¹⁶

A polyurethane coated fabric with bonded seams may be used as a barrier to protect the mattress from disinfectant-related damage. Laundering the barrier can eliminate issues with insufficient removal of pathogens from the patient surface and residual disinfectant on the mattress cover. A barrier provided by Trinity Guardion has been shown to decrease C diff infections (CDIs) in long-term acute care hospitals in the United States.¹⁷ The goal of the current study was to determine the efficacy of a commercial laundry process in reducing bacterial and spore contamination on the barrier.

Materials and Methods

Microorganism and growth conditions

Three suspensions of microorganisms were produced. The first one contained methicillin-resistant Staphylococcus aureus (ATCC 33592), Pseudomonas aeruginosa (ATCC 15442), K pneumoniae (ATCC 10031), and Escherichia coli (ATCC 11229). The organisms were propagated on Tryptic Soy Agar with 5% Sheep Blood, incubated, isolated in Tryptic Soy Broth (TSB), incubated, centrifuged, and the pellet resuspended in 10-mL TSB. Initial organism concentrations were determined by performing 10-fold serial dilutions of each organism. Dilutions were plated in triplicate and incubated at 35°C for 48 hours. After incubation, colonies were enumerated and recorded as colony-forming unit (CFU)/plate with triplicate plates. The mean number was then multiplied by the dilution factor to determine CFU/mL of suspension.

The second suspension contained Mycobacterium terrae (ATCC 15755), which was propagated on M7H10 agar plates, incubated until sufficient growth was observed, harvested with phosphate-buffered solution (PBS), centrifuged, and the pellet resuspended in 5-mL TSB. Initial organism concentrations were determined by performing 10-fold serial dilutions in PBS. Dilutions were plated in triplicate and incubated at 35°C for 2 weeks. After incubation, colonies were enumerated and recorded as CFU/plate with triplicate plates. The mean was then multiplied by the dilution factor to determine CFU/mL of suspension.

The third suspension contained C diff spores (ATCC 43598 Strain 1470 Serogroup F). These were produced using the recommended procedures of the Environmental Protection Agency.¹⁸

Frozen stock was struck to CDC Anaerobic 5% Sheep Blood Agar (CABA) media plates, incubated anaerobically for 48 hours at 36°C, isolated in Reinforced Clostridial Medium (RCM), incubated anaerobically at 36°C for 24 hours, spread plated on CABA plates, verified for confluent lawn growth, and incubated anaerobically at 36°C for 10 days. Spore formation was verified and spore-to-vegetative cell ratio was estimated at more than 90% spores. Spores were harvested with PBS-T. After 3 wash cycles of vortex, centrifuge, and resuspension, the suspension was heat treated at 65°C for 10 minutes and evaluated for the spore-to-cell ratio, estimated at 90% or higher. Spore count of at least 10⁸ was verified after plating 10-fold serial dilutions in PBS-T and incubating. Spore suspension was purified using a density gradient medium, followed by 3 wash cycles of vortex, centrifuge, and resuspension with PBS-T. The final spore suspension was evaluated for the spore-to-cell ratio, estimated to be at least 95%. Viable spore count of at least 10⁷ was verified after plating 10-fold serial dilutions in PBS-T and incubating at 36°C for 24 hours. Based on the enumeration, the final suspension was diluted with PBS-T to achieve a desired concentration of 5.0 × 10⁸ spores/mL. Using 10-fold serial dilutions, the final spore concentration was verified to be at least 2.0 × 10⁸ spores/mL.

Soil urine and blood load on barrier

Representative bodily fluids and soils of the health care patient were included to provide additional challenge to the wash load. The soil load was a combination of Hucker’s and Miles soils intended to simulate fecal material and provide the protein and hemoglobin elements of blood.^19,20 To maintain proper consistency, the distilled water in Hucker’s was replaced by the Miles’ rabbit blood/saline mixture and bovine serum. The ink was eliminated from Hucker’s because color and staining properties were not relevant. The soil contained 10 g each of peanut butter, butter, flour, lard, dehydrated egg yolk, plus evaporated milk (15 mL), saline (3 mL), bovine serum (12 mL), dried milk powder (7.2 g), and rabbit blood mixed 1:1 with 0.85% saline (12 mL).

A wash load included 1 test barrier and 11 ballast barriers. Figure 1 shows the distribution of soil, urine, and blood for each wash load. The test barrier and all ballast barriers each received 3.0 g of soil. Three ballast barriers each received an additional 33.3 g. The test barrier and 5 ballast barriers received 100.0 mL of urine each. The test barrier and 2 ballast barriers each received 30.0 mL of rabbit’s blood. All were allowed to dry for at least 24 hours.

Figure 1.

Application of spores, bacteria, soil, and urine on bed barriers.

Inoculation of test barrier

Organisms were applied to test barrier seams, which are the most difficult-to-clean areas. Prior to application, the C diff spore suspension was mixed with a bovine serum organic soil prepared according to EPA MLB SOP MB-31, combining 5% bovine serum albumin (BSA) stock, 7% yeast extract, 20% mucin stock, and 68% PBS spore suspension.²¹ A 1.0-mL aliquot of each suspension was dispensed over a separate 4” × 4” inoculation area (Figure 1). The test microorganisms were spread uniformly over the area using a sterile spreader. The inoculum was allowed to air dry for at least 2 hours and was visibly dry before testing.

Testing

Each test barrier was laundered and evaluated separately. There were 3 test barriers, 1 positive control, and 1 negative control that were in new condition, and 3 test barriers, 1 positive control, and 1 negative control that had undergone 200 laundry cycles (to represent end-of-life). Positive controls were inoculated in the same manner as test barriers, but not laundered. Negative controls were not inoculated and laundered.

For each test, 1 test barrier and 11 ballast barriers were laundered in a washer/extractor using laundry chemicals and water at the temperatures listed in Table 1. All barriers were dried at 71°C (160°F).

Table 1.

Laundry process using a Milnor 30015M6J; 35-lb capacity.

Step	Operation	Duration (minutes)	Min water temp (°F)I	Water level	Chemical	Min chemical volume (fl oz/100 lb)
1	Flush, 2 way	2	140	High	Detergenta	1
2	Break/detergent, 2 way	8	160	Low	Alkalib	4
					Detergenta	2
3	Bleach, 2 way	8	160	Low	12.5% chlorine bleachc	6 II
4	Rinse, 2 way	2	140	High	–	–
5	Rinse, 2 way	2	120	High	–	–
6	Rinse, 2 way	2	100	High	–	–
7	Sour, 2 way	4	90	Low	Souring agentd	1
8	Low Spd extract	6	Modify end of cycle for optimum water removal III
	Total time	34

^ISpecified temperature must be achieved and maintained for at least 2 minutes.

^IISpecified volume for bleach is the recommended starting point. Adjust to achieve a chlorine concentration between 125 and 150 ppm for the typical wash load.

^IIIRepositioning the wash load numerous times with the drain open is most effective at eliminating trapped water and reducing drying time.

^aPulse Advance—Gurtler Industries, Inc.; South Holland, IL 60473 US.

^bPulse Ultra—Gurtler Industries, Inc.; South Holland, IL 60473 US.

^cSodium Hypochlorite—Gurtler Industries, Inc.; South Holland, IL 60473 US.

^dNDT Sour—Gurtler Industries, Inc.; South Holland, IL 60473 US.

Residual organism determination

After laundering, the 4” × 4” inoculated area was wiped 3 times up and down and 3 times left and right with moderate hand pressure using a sterile gauze pad wetted with 10 mL of sodium thiosulfate to neutralize any residual disinfectant and remove viable microorganisms. The gauze pad was placed in a sterile Stomacher bag with 90 mL of neutralizer. The bag was homogenized by stomaching and serial dilutions were prepared. The dilutions were plated and incubated as previously described. After incubation, colonies were enumerated and recorded as CFU/plate. The count was totaled for each dilution to calculate the total microorganisms recovered. Triplicate plates were multiplied by the dilution factor, and the mean was then reported as the remaining CFU/test barrier. This entire process was repeated for each microorganism type or group and each of the test barriers and controls.

Study controls

Positive controls were inoculated in the same manner as test barriers, but not laundered. These controls were used to verify organism recovery efficiency and to establish the total viable CFUs on the test barriers before laundering. The recovery target of at least 10⁷ CFUs for each test organism was met.

Negative controls were not inoculated, but laundered to determine if any relevant CFUs were picked up in the laundering process. Negative controls were evaluated after all 3 test barriers were laundered and again after organism recovery. The recovery target of less than 100 CFU/mL for each test organism was met.

Wash water was sampled at the drain by aerobic plate count after completion of the bleach portion of the wash cycle (step 3). The acceptance criteria of less than 10 CFU/100 mL were met.

Total suspension viability

To verify the viability of the inoculum over the testing phase, the inoculum was enumerated at the start and end of the testing phase. Inoculum populations were determined by preparing serial dilutions of the challenge organism suspension in triplicate, plating, and incubating. Colonies were enumerated and recorded as CFU/plate to determine surviving organisms. The mean count for the triplicate plates was then multiplied by the dilution factor to calculate the microbial population (CFU/mL) of the control suspension. The target of less than 0.5 log variance from start to finish was met.

Neutralizer evaluation

Testing ensured that no components of the neutralizing procedure exerted an inhibitory effect on the test microorganisms according to ASTM International standards.²² Testing included a neutralizer effectiveness test, neutralizer toxicity test, and organism viability test, and all met the target of less than 0.2 log₁₀ difference between initial and final plate counts.

Organism recovery efficiency

Efficiency of organism recovery was validated by sampling the positive control 4 times. Recovery efficiency was above 93% in all cases except for 85.7% recovery on the end-of-life test sample for the mixed organism suspension.

Statistics

All statistics were performed using SPSS version 24 software (IBM, Armonk, NY). The significance was set at 0.05 for all tests. Counts of the microorganisms before and after washing were compared using the Mann–Whitney U test. Each investigation was repeated in triplicate both before inoculation and after washing.

Human studies

This study did not involve human participants.

Results

After laundering, no pathogenic microorganisms were detected on any of the test barriers (Table 2) or negative controls. No C diff spores were detected on new barriers or the 3 end-of-life barriers (>6.0 log₁₀ cfu reduction). The 3 new barriers and the 3 end-of-life barriers inoculated with mixed pathogenic bacteria all had no detected bacteria (>6.9 log₁₀ cfu reduction). The 3 new barriers and the 3 end-of-life barriers that had been contaminated with M terrae all had no detected bacteria (>6.9 log₁₀ cfu reduction).

Table 2.

Summary of test results.

Challenge organism	Bed barrier prelaundering (number of laundry cycles)	Inoculum log10 cfu/mL (±SD)	Before laundering positive control log10 cfu/mL	After laundering All 3 replicates log10 cfu/mL	Log10 reduction From inoculum (positive ctrl)	P value From inoculum
Clostridioides difficile spores (ATCC 43598)	0	7.7518 (0.4572)	7.5612	<1.00000a	>6.75 >6.56	.034
	200	7.5188 (0.7213)	7.0202	<1.00000a	>6.51 >6.02	.034
Mycobacterium terrae (ATCC 15755)	0	8.4014 (0.1738)	7.8971	<1.00000a	>7.40 >6.90	.034
	200	8.2007 (0.1738)	7.9371	<1.00000a	>7.20 >6.94	.034
Escherichia coli (ATCC 11229) Pseudomonas aeruginosa (ATCC 15442) Staphylococcus aureus MRSA (ATCC 33592) Klebsiella pneumoniae (ATCC 10031)	0	8.1003 (0.1738)	7.9749	<1.00000a	>7.10 >6.97	.034
	200	9.2498 (0.1680)	7.9068	<1.00000a	>8.25 >6.91	.037

Abbreviations: cfu, colony-forming unit; MRSA, methicillin-resistant Staphylococcus aureus.

^aNo CFUs detected on any test barrier after laundry process.

Each suspension was tested prior to inoculation onto the test barrier, and there were greater than 7.5 log₁₀ cfu/mL of each of the microorganisms in the suspension. All positive controls had greater than 7.0 log₁₀ cfu of microorganisms in the test area, and all negative controls had no detectable growth after laundering.

Discussion

The results indicate a commercial laundry process can remove all pathogenic bacteria and C diff spores from the barrier. This represents greater than 6 log₁₀ reduction in microorganisms, which is far greater than the 1 to 2 log₁₀ cfu reductions seen with manual cleaning using chemical disinfectants.

The laundry process provides detergent, bleach, agitation, and repeatability. These elements allow bacteria and spores to be physically separated from the barrier surface. The chlorine works to kill residual organisms. Multiple rinse cycles allow the microorganisms to be removed from the washing machine. Further study could determine the contribution of these various elements to the end result.

The manual process of cleaning hospital mattresses and beds used in most hospitals is typically a 1-step process, despite being off-label use of the disinfectant and the manufacturer’s multistep instructions for cleaning and disinfection. It is not surprising this process fails to reduce the bacterial counts more than 1-2 log₁₀ cfu.^6-11 Multiple studies have shown that if the previous patient had been infected, or even colonized with bacteria, there is a marked increased risk of the patient getting an HAI.^23-25 Mattress covers are soft hygroscopic materials, and not only will a 1-step process fail to disinfect, it may also leave residual chemical on the surface and damage the material. Many mattress manufacturers recommend a multistep cleaning process, including precleaning, cleaning with mild detergent, rinsing, disinfecting, rinsing again, and inspecting.^26-28 Multiple steps can result in improved cleaning and disinfection and protect the mattress cover material by removing the chemical disinfectant. However, a multistep process with rinses can quadruple bed reprocessing time compared with the 1-step process. In the United States, many hospitals routinely maintain occupancy rates close to 100%, which puts pressure on the environmental services workers to clean the room quickly.

The results of this study must be interpreted in light of its limitations. The study was relatively small sample size. Only 3 barriers were used for each condition (new and after 200 launderings). However, all 6 test barriers had no residual bacteria or spores on them after the laundry process. This study uses a specific laundering process. Different laundry processes and chemicals may yield different results.

Conclusions

In this investigation, laundering using a commercial washer/extractor was able to remove all pathogenic bacteria and C diff spores from a barrier. This may offer a better method to prevent HAIs than currently recommended multistep process of manual cleaning of the mattress and bed deck.