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The Potential for Airborne Dispersal of Clostridium difficile from Symptomatic Patients

  1. Emma L. Best1,
  2. Warren N. Fawley1,
  3. Peter Parnell1, and
  4. Mark H. Wilcox1,2
  1. 1Microbiology Department, Old Medical School, Leeds General Infirmary, Leeds Teaching Hospitals National Health Service Trust, and University of Leeds, Leeds, United Kingdom
  2. 2University of Leeds, Leeds, United Kingdom
  1. Reprints or correspondence: Prof Mark H. Wilcox, Microbiology Dept, Old Medical School, Leeds General Infirmary, Leeds LS1 3EX, West Yorkshire, United Kingdom (mark.wilcox{at}leedsth.nhs.uk).
 
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Abstract

Background. The high transmissibility and widespread environmental contamination by Clostridium difficile suggests the possibility of airborne dissemination of spores. We measured airborne and environmental C. difficile adjacent to patients with symptomatic C. difficile infection (CDI).

Methods. We conducted air sampling adjacent to 63 patients with CDI for 180 h in total and for 101 h in control settings. Environmental samples were obtained from surfaces adjacent to the patient and from communal areas of the ward. C. difficile isolates were characterized by ribotyping and multilocus variable-number tandem-repeat analysis to determine relatedness.

Results. Of the first 50 patients examined (each for 1 h), only 12% had positive air samples, most frequently those with active symptoms of CDI (10%, vs 2% for those with no symptoms). We intensively sampled the air around 10 patients with CDI symptoms, each for 10 h over 2 days, as well as a total of 346 surface sites. C. difficile was isolated from the air in the majority of these cases (7 of 10 patients tested) and from the surfaces around 9 of the patients; 60% of patients had both air and surface environments that were positive for C. difficile. Molecular characterization confirmed an epidemiological link between airborne dispersal, environmental contamination, and CDI cases.

Conclusions. Aerosolization of C. difficile occurs commonly but sporadically in patients with symptomatic CDI. This may explain the widespread dissemination of epidemic strains. Our results emphasize the importance of single-room isolation as soon as possible after the onset of diarrhea to limit the dissemination of C. difficile.

Clostridium difficile infection (CDI) is a major burden to health care facilities [1], with increasing rates since 2002 in the United States [2], Canada [3–5], and Europe [6, 7]. C. difficile is transmissible between hospitalized patients, and control measures to limit cross-infection are part of routine practice. Pragmatically, it is desirable to nurse patients with CDI in isolation, although there is a lack of robust evidence to support the utility of single rooms in preventing transmission. However, limited availability of single rooms in some settings can lead to the frequent management of CDI cases in open wards [8, 9]. With the recognition and emergence of virulent strains associated with CDI outbreaks, such as ribotype 027/NAP1 [1–7], it has become increasingly important to determine how transmission is occurring and to establish effective interventions to minimize these risks.

It has been estimated that a patient with CDI can excrete between 1×104 and 1×107 of C. difficile per gram of feces [10]. C. difficile spores may be resistant to disinfectants and can survive for months or years on contaminated surfaces [11–14]. Environmental contamination with C. difficile spores occurs at as many as 34%–58% of sites despite cleaning, with surfaces of fomites being most frequently contaminated [15–17]. Crucially, the hands of health care workers are significantly more likely to be positive for C. difficile if the environment is heavily contaminated with the bacterium [18].

There are few data to substantiate the risks associated with airborne transmission of C. difficile in hospitals. A number of studies have alluded to the possibility that C. difficile could potentially spread through the air [15, 19–22]. We aimed to determine the extent of C. difficile contamination in ward environments by recovery of C. difficile from air and environmental surfaces in the immediate vicinity of patients with symptomatic CDI. We aimed to establish an epidemiological link between patients and their surroundings, using highly discriminatory DNA fingerprinting, as well as the relationship between near-patient activity and C. difficile aerosolization.

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Methods

Organization of Air Sampling

Approval for the study was obtained from the Leeds Teaching Hospitals' Research Committee. The first phase of the investigation (6 months) comprised air sampling (total, 50 h) for 1 h adjacent to 50 patients with confirmed CDI (including 13 in general medical wards, 25 in elderly care wards, and 12 in the C. difficile ward, of whom 43 were in single rooms and 7 were in multioccupied bays). All air was maintained by standard ventilation. The second and third phases of sampling (4 months) involved 10 h of air sampling over 2 days (total, 130 h for proven CDI cases [see below]) and 40 environmental samples per patient in elderly care wards and the C. difficile ward; 19 of 20 patients in these phases were in single rooms. The second phase of sampling comprised 10 patients with CDI identified by nursing staff as having suspected cases (not laboratory confirmed). The third phase of sampling focused on 10 patients with symptomatic (cytotoxin-positive) CDI.

Control air sampling consisted of the following. During phase 1, 11 air samples (11 h) were obtained adjacent to patients without a history or symptoms suggestive of CDI. During phase 2, 7 of the 10 patients with suspected CDI proved to be cytotoxin negative and were therefore counted as control tests (70 h). During phase 3, air sampling was conducted adjacent to a patient without a history of CDI (10 h) and in the corridor of the same ward (10 h). Air sampling totaled 180 and 101 h for CDI case patients and control subjects. During sampling, we observed and recorded ongoing activities that could be associated with marked air disturbance (eg, bed changing and frequency of visitors). Routine environmental cleaning was done each morning, using a detergent/chlorine (1000 ppm) agent.

Air Sampling

Air was collected using 1 of 2 AirTrace Environmental portable samplers (Biotrace; Microbial Contamination Control). Air sampling was conducted as close as practically possible to the patient's bed, via a 2-m Tygon tube (Saint-Gobain) placed within 1 m at the foot of the bed. As the air entered the sampler (28.3 L/min), it was forced through a fine slit (44×0.152 mm) at a velocity of 70 m/s, thereby causing particulate matter (minimum size, 0.4 µm) to impact on a C. difficile selective agar plate. Plates (140-mm diameter) contained Brazier's cycloserine-cefoxitin-egg yolk agar (Bioconnections) supplemented with 5 mg/mL lysozyme (CCEYL) (not prereduced) [23]. The plate rotated constantly; thus, after culture the location of the colonies represented the time of recovery from air, making it possible to link it to activities near to the sampler. Plates were transported to the laboratory and incubated anaerobically (37°C for 48–72 h). After each sampling session or day, the machine was cleaned externally and internally with a sporicidal disinfectant (Trigene; Medichem).

Environmental Surface Sampling

Initial in vitro tests for determining the efficiency of Polywipe sponges.Polywipe sponges (Medical Wire & Equipment) were initially examined as a pilot to this investigation to determine their efficiency for recovery of C. difficile from environmental surfaces. In vitro tests (in triplicate) were conducted using 200-µL aliquots of diluted spore suspensions (2.4–2.4×105) spread onto an aluminium surface (30×15 cm) and left to dry for 1 h. Duplicate test-seeded areas were sampled using both standard environmental swabs and sponges. Each sponge or swab tip was placed into a Stomacher bag (Seward) with 50 mL of Ringer solution and processed for 30 s. Aliquots were cultured on CCEYL and anaerobically enriched in cooked-meat fastidious anaerobe broth (E&O Laboratories) at 37°C for 48 h.

Patient environment sampling.Following the pilot study, sponges were used for environmental sampling (Table 1). After sampling, 50 mL of Ringer solution was added to each sponge, followed by 30-s processing in a Stomacher bag. Liquid from the bag was passed through a 0.45-µm filter (Millipore), which was then placed in 10 mL of cooked-meat broth and subcultured on CCEYL, as described above.

Figure 1
Figure 1

Line indicating the total number of Clostridium difficile colonies recovered at various times throughout the day (total of 10 patients tested for 2 days). The number of patients the colonies were isolated from is indicated in parentheses.

Table 1
Table 1

Detail of 20 Surfaces Tested during Environmental Sampling Using Polywipe Sponges (Surface Area, ∼30 cm2)

Culture of Patient Fecal Samples

Patient fecal samples (pea-sized aliquots) were alcohol-shocked by immersion in 1 mL of 50% ethanol solution. After being vortexed for 10 s and left to stand at room temperature for ⩾1 h, samples were cultured on CCEYL plates, as described above.

Polymerase Chain Reaction Ribotyping and Multilocus Variable-Number Tandem-Repeat Analysis

Polymerase chain reaction ribotyping was performed on all C. difficile isolates as described previously [24]. Multilocus variable-number tandem-repeat analysis (MLVA) was conducted as described elsewhere [25], using 7 loci (A6, B7, C6, E7, F3, G8, and H9). Fragments were analyzed using GeneMapper software (version 4.0; Applied Biosystems), and copy numbers were determined. The summed absolute difference between 2 MLVA-typed isolates is the calculated summed tandem-repeat difference (STRD) at all 7 loci [26]. MLVA types with a STRD ⩽2 were indicative of a high degree of genetic relatedness among C. difficile isolates.

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Results

Polywipe sponge in vitro tests.Sponges were significantly more effective than swabs (P=.006) at recovering C. difficile from surfaces (Table 2). In tests comprising 2400 colony-forming units (CFUs) spread on a test surface, a recovery of 52% with sponges versus zero with swabs was achieved. To increase the detection limit further (2 CFUs), an enrichment step was added, on the basis of previous experience with recovering C. difficile from environmental sites [23]. Sponges also allowed sampling of larger surface areas, and so these were used in preference to swabs during phases 2 and 3.

Table 2
Table 2

Mean Clostridium difficile Counts from In Vitro Surface Recovery Tests for Polywipes and Swabs

First-phase sampling of 50 patients with confirmed CDI.Of the first 50 patients examined (1 h), only 6 (12%) had positive cultures from the air sampling. There was a trend toward there being more positive air samples from patients with active diarrheal symptoms, compared with those without diarrheal symptoms (10% vs 2%; P=.1). Of the 5 symptomatic patients with positive air samples, 2 were in beds on different 6-bedded bays (fully occupied), and 3 were in single rooms. During air sampling, cleaning and bed making were taking place close to 2 of the positive patients. No C. difficile was recovered from any of the control air samples (n=11).

Second-phase air and environmental sampling of symptomatic patients with suspected but unconfirmed CDI.Results from phase 1 suggested that airborne dissemination of C. difficile spores may be occurring before laboratory confirmation of CDI was obtained. Of 10 patients identified by nursing staff as having suspected CDI, only 3 proved to have laboratory confirmed cases. Patient U4, who had a positive air sample, was in a single room and underwent air sampling on 15 December 2008 and 16 December 2008, with CDI confirmed on 16 December 2008. C. difficile colonies were recovered on 3 occasions (day 1, at 1115, 1130, and 1315), corresponding to ward cleaning at 1130 and to curtain closure around the bed at 1315. Ribotyping and MLVA confirmed that indistinguishable isolates were recovered from the patient and air at each time point (ribotype 106; MLVA profile 23-13-23-2-6-4-2). Patients U2 and U5 were also confirmed to be positive for CDI, but no positive air or environmental samples were obtained. The remainder of the patients (n=7) were confirmed to be negative for cytotoxin and were thus considered as control (albeit diarrheal) patients. A positive air sample was collected from one of these control patients (U8), and positive environmental samples were collected from patient U6 (commode) and from patient U9 (bed, floor, table, sink, and the ward storeroom handle). C. difficile with the same ribotype and MLVA type (027; 31-22-17-12-5-8-2) was obtained from the bed, floor, table, and sink, but C. difficile with a different ribotype and MLVA type (106; 24-14-22-2-6-4-2) (STRD, >10) was recovered from the storeroom door.

Third-phase air and environmental sampling of symptomatic patients with confirmed CDI.Of the 10 patients tested (10 h), 7 had at least 1 positive air sample, 4 on multiple occasions. The most common times when C. difficile was recovered from the air corresponded with activity close to patients (Figure 1). Between 1000 and 1100, a drinks delivery occurred for patients, which corresponded to the recovery of C. difficile from 2 patients. The peak at 1145 corresponded with ward rounds between 1100 and 1200. The second peak comprising a total recovery of 8 colonies (from 4 patients) corresponded to lunch delivery (1200) and visiting time (1200–1400).

In total, 346 environmental surface samples were obtained during this phase, of which 10% yielded C. difficile (from 9 of 10 patients). For the single patient without a positive environmental sample, C. difficile was recovered from the air. The highest levels of recovery of C. difficile were from surfaces closest to the patients and the areas frequently handled, including the patients' bed, bedside table, sink, and bin (6 or more isolations). There were fewer (<2) positive environmental samples from infrequently touched surfaces.

Six patients had C. difficile-positive environmental and air samples (Table 3). For 3 patients, C. difficile recovered from the air and at least 1 environmental sample had identical MLVA types. For the other 3 positive patients, the C. difficile recovered from the air in each environment was considered to be highly related to at least 1 environmental sample obtained within the same environment (either a single- or double-locus variant, with a STRD ⩽6). It was also possible to confirm an epidemiological link for the C. difficile isolates from air, diagnostic fecal samples, and environmental surfaces. For example, for patient C1 the C. difficile isolates from feces, air (1045 and 1050), and a table were all ribotype 027, with highly related MLVA types and 6 identical loci (31-22-15-12-5-10-2 and 31-22-15-12-5-8-2; a single-locus variant with a STRD of 2). C. difficile was not recovered from 20 h of control air samples.

Table 3
Table 3

Molecular Testing of Patient Samples and Phase 3 Positive Air and Environmental Samples for Symptomatic Patients with Confirmed Clostridium difficile Infection

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Discussion

Airborne transmission and environmental contamination of C. difficile was first suspected in hamster experiments [27]. Studies reported significant contamination of objects in the immediate environment and suggested that airborne cross-contamination appeared to be less important than that from contact surfaces [21, 27]. Fekety et al [22] used a slit-impaction air sampler and failed to isolate C. difficile from the air around patients with CDI but demonstrated extensive environmental and hand contamination. Roberts et al [19] recovered C. difficile from air using a cyclone air sampler in a hospital but did not recover C. difficile from associated environmental surfaces. Our study appears to be the first to recover C. difficile from the air and environment within the same time period and provide confirmation of a link between isolates.

We have shown that C. difficile is commonly (from 7 of 10 patients intensively studied) but sporadically present in the air close to symptomatic patients with CDI. By MLVA, we confirmed the presence of indistinguishable or very highly related strains of C. difficile in the environment, patient fecal specimens, and air. Several studies have confirmed the utility of MLVA for discrimination within C. difficile ribotypes [25, 28], including ribotype 106 (authors' unpublished data). Thus, our findings help explain the widespread dissemination of C. difficile in the hospital environment, including to infrequently touched or cleaned sites [11–13, 15, 16]. For example, we previously showed that 69% of infrequently touched (“high dust”) surfaces were positive for C. difficile in an elderly medical ward within 6 months of ward opening [29]. We emphasize that without prolonged sampling of air we would have underestimated the sporadic nature of airborne C. difficile dispersal. This may explain the failure of some investigators to detect C. difficile in air [21, 22, 27].

We detected airborne C. difficile most commonly during periods of activity, particularly around the busy lunchtime period. These observations suggest that the air within the patient's immediate environment is contaminated with C. difficile spores either directly from symptomatic patients or from environmental surfaces and that people movement, including the opening and closing of doors, contributes to the circulation and dispersal of airborne C. difficile. It has been demonstrated previously that areas associated with much air movement, such as air vents, are contaminated with C. difficile [29, 30]. These findings have implications for cleaning practices. Unless cleaning is done frequently around symptomatic patients with CDI, including infrequently touched places, reaccumulation of C. difficile will occur on surfaces via the air. Disturbance of already-contaminated articles, such as the bin or bed linen, may contribute to spore aerosolization. It has been demonstrated previously that bed linen can be contaminated [31], and during this study we demonstrated that 3 of 30 bed curtains were culture positive for C. difficile on a ward with 2 confirmed CDI cases (authors' unpublished data). Therefore, activities known to liberate particles into the air, such as bed making and curtain drawing [32, 33], as well as contact with these items may contribute to the spread and aerosolization of C. difficile.

The environmental surface sampling results were consistent with previous studies showing that frequently touched areas are most often C. difficile positive [17, 31, 34]. C. difficile was not recovered from the least-touched areas, such as the light. The patient room door handles were very infrequently found to harbor C. difficile, which presumably reflects the frequent use of hand hygiene practices immediately before entering or leaving rooms. Conversely, C. difficile was recovered from sluice room door handles, likely reflecting contamination by staff disposing of feculent material. It might be expected to recover C. difficile from the patients' communal bathrooms [17, 31, 34]. However, such facilities were unlikely to be frequented by patients with CDI during this study, because they tended to use en-suite rooms and/or their own commodes.

There are some limitations to the present study. C. difficile recovery was generally modest, which may reflect methodological problems or a genuine low environmental microbial burden. The air sampler used was reliant on a slit-to-agar impaction method. A similar study [19] used a machine that recovered airborne material directly into solution but recorded airborne C. difficile counts that were orders of magnitude greater than our results. It is possible that positioning of the air sampler next to a toilet may have partly explained the higher airborne counts of C. difficile [19]. Because we sampled air close to patients for 5-h periods, we had to use the machine contained within a soundproof box and collect samples via an extension tube. The tube may have resulted in a loss of particulate matter collected onto the plates. The practicalities of prolonged sampling close to patients may have caused inconsistencies; for instance, during testing the air sampler or tube may have been moved because of visitors or patient care activities. A further difficulty was the timing and extent of symptoms, particularly because our phase 1 data suggested that air contamination by C. difficile was more likely while diarrhea was occurring. Although we had confirmation that patients were C. difficile toxin positive, we had to rely on health care staff to inform us of symptoms, and so sampling likely occurred at different times relative to the onset of CDI. For future studies, it would be useful to conduct air sampling before (this would also increase sampling of control subjects, which was limited in our study) and during the course of CDI to determine the frequency of C. difficile aerosolization in symptomatic patients.

It remains unclear whether the frequent presence of particular strains in health care environments reflects the burden of CDI caused by epidemic types or whether these have enhanced capacity to persist—for example, because of greater sporulation [30, 35, 36]. Nevertheless, our results suggest that there is a clear risk for C. difficile contamination via the air, particularly in patients with active CDI symptoms. The efficacy of these approaches as a control mechanism for CDI remains unproven. By contrast, the results of the present study do justify the use of single rooms for patients with suspected or proven CDI, even when such resources are limited [8]. In particular, we believe our findings underscore the importance of early patient isolation, as soon as possible after the onset of diarrhea and before laboratory diagnosis of CDI is confirmed. Allowing even a few hours before patient isolation or the wait until laboratory diagnosis is obtained, even with rapid tests, may not be adequate to prevent environmental dissemination of C. difficile via the air. Such a mechanism would at least partly explain the rapid spread and large outbreaks of CDI typified by epidemic strains, such as C. difficile ribotype 027 [1–7]. Recognition of the risk of airborne dissemination provides an opportunity to reduce transmission, especially of epidemic C. difficile strains.

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Acknowledgments

Polywipe sponges were provided free of charge by Medical Wire & Equipment (Corsham, Wiltshire, United Kingdom) for evaluation as part of this study.

Financial support.Study B(07)08, Estates and Facilities Division, Research and Development Fund, Department of Health, United Kingdom.

Potential conflicts of interest.All authors: no conflicts.

  • Received November 18, 2009.
  • Accepted January 6, 2010.
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  1. Clin Infect Dis. (2010) 50 (11): 1450-1457. doi: 10.1086/652648
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