Assessment of the Overall and Multidrug-Resistant Organism Bioburden on Environmental Surfaces in Healthcare Facilities

Abstract

OBJECTIVE.

To determine the typical microbial bioburden (overall bacterial and multidrug-resistant organisms [MDROs]) on high-touch healthcare environmental surfaces after routine or terminal cleaning.

DESIGN.

Prospective 2.5-year microbiological survey of large surface areas (>1,000 cm²).

SETTING.

MDRO contact-precaution rooms from 9 acute-care hospitals and 2 long-term care facilities in 4 states.

PARTICIPANTS.

Samples from 166 rooms (113 routine cleaned and 53 terminal cleaned rooms).

METHODS.

Using a standard sponge-wipe sampling protocol, 2 composite samples were collected from each room; a third sample was collected from each Clostridium difficile room. Composite 1 included the TV remote, telephone, call button, and bed rails. Composite 2 included the room door handle, IV pole, and overbed table. Composite 3 included toileting surfaces. Total bacteria and MDROs (ie, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci [VRE], Acinetobacter baumannii, Klebsiella pneumoniae, and C. difficile) were quantified, confirmed, and tested for drug resistance.

RESULTS.

The mean microbial bioburden and range from routine cleaned room composites were higher (2,700 colony-forming units [CFU]/100 cm²; ≤1–130,000 CFU/100 cm²) than from terminal cleaned room composites (353 CFU/100 cm²; ≤1–4,300 CFU/100 cm²). MDROs were recovered from 34% of routine cleaned room composites (range ≤1–13,000 CFU/100 cm²) and 17% of terminal cleaned room composites (≤1–524 CFU/100 cm²). MDROs were recovered from 40% of rooms; VRE was the most common (19%).

CONCLUSIONS.

This multicenter bioburden summary provides a first step to determining microbial bioburden on healthcare surfaces, which may help provide a basis for developing standards to evaluate cleaning and disinfection as well as a framework for studies using an evidentiary hierarchy for environmental infection control.

Healthcare-associated infections (HAIs) are a significant cause of morbidity and mortality in the United States. A 2011 multistate point-prevalence survey recorded an estimated 722,000 HAIs in 648,000 hospitalized patients resulting in ~75,000 patient deaths.¹ Prevention efforts have recently focused on the role of the physical environment in the transmission of pathogens causing HAIs. High-touch, noncritical hospital surfaces (eg, bed rails, overbed tables, and IV poles) are commonly contaminated with pathogens, and many have been linked to outbreaks in hospitals and long-term care facilities (LTCFs).^2–12

Most published studies assessing microbial contamination on environmental surfaces in healthcare facilities have been qualitative, reporting only the proportion of positive samples.^{3–5,9,10,13} The few studies reporting quantitative results focus either on the amount of overall contamination or contamination by specific pathogens, usually not both.^14–21 Microbial bioburden (MB), when reported, is only from individual surfaces, such as bed rails, or is reported in relation to cleaning method or product efficacy testing.^21–26 These studies can be difficult to compare because they often use different sampling methodologies or reporting _units.^{15,17,19–22,27}

The lack of consistent quantitative contamination data makes it difficult to correlate bioburden reductions when assessing cleaning procedures or disinfection products. Current CDC guidelines contain no recommended standard MB levels.²⁸ More than a decade ago, benchmark standards were proposed for high-touch surfaces in UK hospitals to correlate cleanliness and MB.^29,30These standards of <5.0 colony-forming units (CFU)/cm² or <2.5 CFU/cm² are of limited use because the dip-slide sampling method must be used, which limits the sampling to flat surfaces of <12 cm².^12,29–33 Without knowing the true MB on hospital environmental surfaces, it can be difficult to assess achievable bioburden concentrations that will impact patient safety. The main objectives of our study were to use standardized, large-area (>1,000 cm²), sponge-wipe sampling and laboratory processing procedures to determine typical MB (overall bacterial and specific multidrug-resistant organisms [MDROs]) on high-touch, noncritical, healthcare environmental surfaces after routine or terminal room cleaning across a number of facilities.

METHODS

Study Design

This prospective study was conducted between January 2011 and July 2013 in 9 acute-care hospitals and 2 LTCFs in 4 states. Rooms of patients on contact precautions for MDROs (eg, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Acinetobacter baumannii, Klebsiella pneumoniae, and Clostridium difficile) were sampled after either routine or terminal cleaning.

Environmental Sampling

Samples were collected using environmental sponge-wipes (3M Sponge-Stick with neutralizing buffer; 3M, St. Paul, MN). To best evaluate contamination of the room as a whole, a preliminary study was performed to identify high-touch sites that could be combined as composite samples. Up to 12 high-touch sites (surface area ≤1 m²) were sampled in patient rooms from 9 healthcare facilities. Each site included IV pump and/or pole, television remote, overbed table, telephone, door handles, call bell, bed rails, supply cart, bathroom hand rail, and toilet handle.

Based on the preliminary data, we developed 3 composites that included items common in the patient rooms of all participant hospitals and LTCFs. The sites sampled as part of each composite were assigned based on a maximum total sampling surface area of 2,258.06 cm² per composite and were composed of 1 large surface-area site (bed rails or overbed table) and 2–3 smaller sites. One sponge wipe was used for each composite. Sites were assigned as follows: composite 1 (C1) included bed rails, television remote, call button, and telephone; composite 2 (C2) included overbed table, IV pole, and inside room door handle; and composite 3 (C3) included either the portable commode (grab bars and seat), bedpan (bathroom door handle, toilet flush handle, rinse spout handle, and seat), or bathroom (door handle, flush handle, and grab bar) in rooms of patients with C. difficile. Bathroom sites were included for C. difficile contact precaution rooms to increase the chance of recovering C. difficile.

The healthcare facilities notified samplers when MDRO contact precaution rooms occupied by patients diagnosed with the specificed MDR infections were available for sampling. Samples were collected as soon as possible after routine or terminal cleaning and rooms were of similar design on the same unit or ward. “Routine” was defined as the daily cleaning procedure for patient rooms, and samples were collected when patients were out of the rooms. “Terminal” referred to the cleaning and disinfection procedures that occurred after patients were discharged. Routine and terminal cleaning procedures varied by facility. The exact surface area sampled was recorded for each composite, along with cleaning products used, cleaning and sampling times, and specific patient clinical factors (ie, acute diarrhea, presence of catheters, open wounds, dialysis, etc). For each facility, up to 20 routinely cleaned and 10 terminally cleaned rooms were sampled. Field blank samples were collected before and after sampling each room. Samples were shipped to the Centers for Disease Control and Prevention (CDC) for processing on the day of collection when possible.

Sample Processing and Culture Methods

All samples were processed immediately upon receipt at the CDC. Sponge wipes were expressed in 90mL phosphate-buffered saline containing 0.02% Tween 80 (PBST) using a stomacher. The eluate was then concentrated by centrifugation. The pellets were resuspended in PBST and cultured. To recover and quantify target MDROs, aliquots were cultured on CHROMagar VRE (CHROMagar, Becton Dickinson, San Jose, CA) for VRE, mannitol salt agar (Becton Dickinson) for S. aureus, MacConkey agar (Becton Dickinson) for K. pneumoniae, MDR-Acinetobacter agar (Hardy Diagnostics, Santa Maria, CA) for A. baumannii, and cycloserine cefoxitin fructose agar with horse blood and taurocholate (Anaerobe Systems, Morgan Hill, CA) for C. difficile. Total aerobic (tryptic soy agar with 5% sheep’s blood; Becton Dickinson) and total anaerobic counts (anaerobic blood agar; PathCon Laboratories, Norcross, GA) were determined. All media were inoculated in duplicate and incubated as appropriate. Broth enrichment was used to recover low levels of target MDROs; tryptic soy broth (Becton Dickinson) was used for Gram-negative bacteria; tryptic soy broth with 6.5% NaCl (Becton Dickinson) was used for MRSA and VRE; and cycloserine cefoxitin fructose broth was used for C. difficile.³⁴ Broth cultures were subsequently subcultured on selective media as described above.

Bacterial Identification and Antibiotic Resistance Testing

Identification of suspect bacteria was confirmed using conventional biochemical methods (Vitek 2; bioMeriuex, Marcy-l’Étoile, France). C. difficile identification and presence of toxin genes were confirmed by polymerase chain reaction.³⁵ Antimicrobial susceptibility testing was performed on confirmed isolates using standard protocols (disk diffusion or broth microdilution).³⁶ MDR-A. baumannii isolates were defined using the 3-class-resistant definition.³⁷ K. pneumoniae isolates were considered resistant if they were positive for extended-spectrum β-lactamase (ESBL) by broth microdilution, New Delhi metallo-β-lactamase (bla_NDM-1), or carbapenemase (bla_KPC) by polymerase chain reaction.^36,38

Data Analysis

For the preliminary study, the mean, standard deviation, and distribution (median and empirical distribution function (EDF) using nonparametric Brown-Mood and Kuiper tests) of overall aerobic MB per area sampled (CFU/100 cm²) for each individual site were compared to develop the composite sampling strategy.

Descriptive statistics for overall MB per area sampled and each MDRO were calculated for the main sampling study composites and rooms using univariate analysis. Samples that were only broth positive were assigned a value of 1 CFU in order to be included in the analysis. Overall MB for each sample was calculated from the maximum aerobic or anaerobic colony counts, and total room MB was determined by summing C1 and C2 MB; C3 was not included due to the low number of samples collected. Total room MB was log normalized and compared using the Student t test (P≤.05). SAS statistical software, version 9.3 (SAS Institute, Cary NC) was utilized.

RESULTS

Preliminary Study

Data from the preliminary study of 102 samples from 13 routine cleaned rooms revealed that surfaces with the highest mean bacteria were the room door handles (7,546 CFU/100 cm²), telephone (2,350 CFU/100 cm²), and remote/call button (1, 353 CFU/100 cm²; Online Supplementary Table S1). There was a ~50-fold variation in overall mean aerobic bacteria across the sites, and although MDROs were recovered from several sites, the sites with the greatest bioburden (door handles) yielded no MDROs. The overbed table was the most common MDRO-positive site (53.9%). The number of MDRO-positive sites ranged from 0.0 to 80.0% per room (Online Supplementary Table S2); however, the number of sites sampled per room varied from 4 to 9 sites.

Main Study

During the main study, 375 composite samples were received. After excluding 15 samples due to insufficient information, 360 samples were analyzed. More composites were received from routine cleaned rooms (C1s, 113; C2s, 113; C3s, 16; total= 242) than from terminal cleaned rooms (C1s, 53; C2s, 53; C3s, 12; total= 118). The MB mean and range of composites from routine cleaned rooms (2,700 CFU/100 cm²; ≤1–130,000 CFU/100 cm²) was notably higher than that of composites from terminal cleaned rooms (353 CFU/100 cm²; ≤1–4,300 CFU/100 cm²; Table 1). C1s from routine cleaned rooms had the highest mean MB (3,800 CFU/100 cm²), while C2s from terminal cleaned rooms had the lowest bioburden (244 CFU/100 cm²; Fig. 1).

TABLE 1.

Overall Microbial and Individual MDRO Bioburden Detected in Composite Samples From Routine Cleaned (n =242) and Terminal Cleaned Rooms (n =118)

	Overall MB, CFU/100 cm2		MRSA, CFU/100 cm2		VRE, CFU/100 cm2		MDR-A. baumannii, CFU/100 cm2		K. pneumoniae, CFU/100 cm2		C. difficile, CFU/100 cm2
	Routine	Terminal	Routine	Terminal	Routine	Terminal	Routine	Terminal	Routine	Terminal	Routine	Terminal
Mean	2,700	353	79	0.12	42	1	6	0.01	27	4	1	0.57
SD	11,042	742	852	0.84	204	7	69	0.06	208	48	6	2.9
Range	≤1–30,000	≤1–4,300	≤1–13,000	≤1–8	≤1–1,680	≤1–59	≤1–1,061	0.0–0.7	≤1–2,051	≤1–524	≤1–65	≤1–22

NOTE. MDRO, multidrug-resistant organism; MRSA, methicillin-resistant Staphylococcus aureus; MB, microbial burden; VRE, vancomycinresistant enterococci; MDR, multidrug resistant; CFU, colony-forming units; SD, standard deviation.

FIGURE 1.

Microbial bioburden by composite type from routine and terminal cleaned rooms; mean (standard deviation), routine cleaned composites C1 and C2 (n =113 each), composite 3 (n= 16); terminal cleaned composites C1 and C2 (n= 53 each) and composite C3 (n =12); CFU, colony-forming units.

For each MDRO, recovery from routine cleaned room composites was markedly higher than from terminal cleaned room composites (Table 2). When assessing only the MDRO-positive samples, the highest mean MDRO bioburden was K. pneumoniae from routine cleaned room composites (1,284 CFU/100 cm²) and the lowest was A. baumannii (0.66 CFU/100 cm²) from terminal cleaned room composites (Table 2).

TABLE 2.

Individual MDRO Bioburden Detected in MDRO-Positive Samples from Routine Cleaned and Terminal Cleaned Rooms

	MRSA, CFU/100 cm2		VRE, CFU/100 cm2		MDR-A. baumannii, CFU/100 cm2		K. pneumoniae, CFU/100 cm2		C. difficile, CFU/100 cm2
	Routine	Terminal	Routine	Terminal	Routine	Terminal	Routine	Terminal	Routine	Terminal
n	22	5	45	7	6	1	5a	2b	24	9
Mean	873	3	223	18	222	0.66	1,284	262	11	7
SD	2,760	3	433	21	416	...	765	370	15	8

NOTE. MDRO, multidrug-resistant organism; MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-resistant enterococci; MDR, multidrug resistant; CFU, colony-forming units; n; number of composites positive for MDRO; SD, standard deviation.

^aAll were extended-spectrum β-lactamase positive (ESBL+).

^bOne sample was carbapenemase (bla_KPC) positive and 1 was ESBL+.

MDROs were recovered from 33.9% of composites from routine cleaned rooms with the highest recovery for any MDRO from C3 (43.8%) followed by C1 (34.5%) and C2 (31.9%; Table 3). VRE was the most frequently recovered MDRO from all 3 composites from routine cleaned rooms (18.6% of C1s; 16.8% of C2s; 31.3% of C3s). All 5 target MDROs were recovered from C1s and C2s, but only MRSA, VRE and C. difficile were recovered from C3s. In total, 24 isolates of C. difficile were recovered; however, 50% were recovered from non–C. difficile rooms. Most C. difficile isolates were recovered from C1s (38%) or C2s (54%), very few were recovered from C3s (8%). The 2 isolates recovered from C3s were from C. difficile isolation rooms. In total, 6 A. baumannii isolates were MDR and 6 K. pneumoniae isolates were ESBL producers.

TABLE 3.

Percent of Composites Positive for Given MDRO from Routine Cleaned and Terminal Cleaned Rooms

	Composite 1, % (No.)		Composite 2, % (No.)		Composite 3, % (No.)
MDRO	Routine	Terminal	Routine	Terminal	Routine	Terminal
MRSA	10.6 (12)	7.5 (4)	7.1 (8)	1.9 (1)	12.5 (2)	0
VRE	18.6 (21)	5.7 (3)	16.8 (19)	5.7 (3)	31.3 (5)	8.3 (1)
MDR-A. baumannii	2.7 (3)	1.9 (1)	2.7 (3)	0	0	0
K. pneumoniae	2.7 (3)	3.8 (2)	1.8 (2)	0	0	0
C. difficile	8.0 (9)	7.5 (4)	11.5 (13)	7.5 (4)	12.5 (2)	8.3 (1)
Any MDRO	34.5 (39)	20.8 (11)	31.9 (36)	15.1 (8)	43.8 (7)	16.7 (2)
Total	113	53	113	53	16	12

NOTE. MDRO, multidrug-resistant organism; MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycin-resistant enterococci;MDR, multidrug resistant.

Composites from terminal cleaned rooms were less frequently MDRO positive (17.8%), with the highest recovery from C1s (20.8%), then C3s (16.7%) and C2s (15.1%; Table 3). The individual MDRO recovery was low (<8.3%), and no individual MDRO was consistently recovered. All 5 MDROs were recovered from C1s, but A. baumannii and K. pneumoniae were not recovered from C2s, along with MRSA from C3s. In total, 9 C. difficile isolates were recovered, and 50% were recovered from C. difficile isolation rooms. Only 1 room was positive for C. difficile from a C3 alone; 1 A. baumannii isolate was MDR, 1 K. pneumoniae isolate was an ESBL producer; and another was a carbapenemase producer.

For room-level evaluations of MB and MDRO bioburden, 166 rooms were assessed (113 routine cleaned rooms and 53 terminal cleaned rooms). In routine cleaned rooms, the mean MB (5,373 CFU/100 cm²) and mean MDRO bioburden (302 CFU/100 cm²) were almost 8 and 24 times higher, respectively, than in terminal cleaned rooms (687 and 13 CFU/100 cm²; Figure 2). There was a significant difference between routine and terminal cleaned rooms for recovery of log₁₀ MB (P = .0002).

FIGURE 2.

Overall microbial bioburden and multidrug-resistant organism (MDRO) bioburden for routine and terminal cleaned rooms (composites 1 and 2 summed); mean (standard deviation); CFU, colony-forming units.

MDROs were recovered from 39.8% of rooms (75.8% routine; 24.2% terminal) (Fig. 3). Almost 45% of routine cleaned rooms and 30% of terminal cleaned rooms were positive for an MDRO. MRSA was the most common contact precaution-room type sampled (79%); however, VRE was the predominantly recovered MDRO from all rooms (19.3%) and from routine cleaned rooms (23.9%); C. difficile was the predominantly recovered MDRO from terminal cleaned rooms (11.3%). For all MDROs, except MRSA, more rooms were positive for a discordant MDRO than concordant contact precaution MDRO (Table 4). VRE was recovered more often from discordant contact precaution rooms (n = 20) than from concordant VRE contact precaution rooms (n = 12), and all 5 rooms positive for K. pneumoniae were discordant contact precaution rooms. Multiple MDRO types were recovered from 11 rooms.

FIGURE 3.

Percent recovery of each target multidrug-resistant organism (MDRO) and all MDROs from routine cleaned, terminal cleaned, and all rooms.

TABLE 4.

Summary of MDRO Recovery From Contact Precaution Rooms

MDRO	Rooms Positive for MDRO Recovery	Rooms Positive for Concordanta Contact Precaution MDRO, No. (total contact precaution rooms sampled)b	Rooms Positive for Discordantc Contact Precaution MDRO
MRSA	19	16 (92)	3
VRE	32	12 (51)	20
MDR-A. baumannii	6	1 (3)	5
K. pneumoniae	5	0 (4)	5
C. difficile	25	10 (30)	15

NOTE. MDRO, multidrug-resistant organism; MRSA, methicillin-resistant Staphylococcus aureus; VRE, vancomycinresistant enterococci; MDR, multidrug resistant.

^aMDRO(s) recovered by sampling that matched the patient’s diagnosis which required contact precautions.

^bIn total, 23 patients were on contact precautions for multiple MDROs.

^cMDRO(s) recovered by sampling from room that did not match the MDRO causing the patient to be on contact precautions.

DISCUSSION

We developed a composite sponge-wipe, large-surface-area sampling plan to determine typical bacterial bioburden levels in MDRO contact precaution rooms after routine or terminal cleaning. We found that the mean MB collected from surfaces was 2,700 CFU/100 cm² in routine cleaned rooms and 353 CFU/100 cm² in terminal cleaned rooms. These results present a broad but not representative cross section of current levels of contamination across these facilities.

When we assessed bioburden by composite type, we found that surfaces sampled as part of C1 (ie, bed rails, TV remote, call button, and telephone), which were usually closest to the patient, were often the most contaminated, which is consistent with other studies.^{12,19,22–24} Similar MB was reported by Schmidt et al²³ on plastic bed rails after cleaning (1,112–5,198 CFU/100 cm²). However, while C1s had the highest MB in samples from routine and terminal cleaned rooms, C2s and C3s were within the same magnitude (log₁₀).

When we combined C1s and C2s to determine overall room bioburden, routine cleaned rooms were ~8 times more contaminated than terminal cleaned rooms. This difference is significant and was expected because terminal cleaning procedures are often more thorough and use more efficacious cleaning/disinfection products (eg, bleach or hydrogen peroxide vapor) than routine cleaning procedures. In addition, previous research on cleaned bed rails has shown that bacterial counts can rebound within a few hours of cleaning during ongoing patient care.²⁴ In our study, the time between cleaning and sampling varied for both routine and terminal cleaned rooms.

In this study, we also attempted to determine the bioburden levels of 5 target MDROs. We found that ~40% of the rooms sampled were positive for any MDRO, the majority of which were routine cleaned. Overall, mean MDRO bioburden was low for each target organism; however, even the presence of low amounts of any MDRO on surfaces in patient rooms is cause for concern. While we cannot directly compare MDRO recovery from this study to other published research because facilities and regions have their own unique microbiome, these results do show that MRSA, VRE, and C. difficile are more often found on these heathcare surfaces than A. baumannii and K. pneumoniae. We also found that recovered MDROs often were discordant with the MDRO requiring contact precautions and that many rooms were positive for MDROs different than those for which patients were isolated or for additional MDROs (Table 4). The presence of MDROs other than those for which patients were isolated may be due to undetected carriage of the current patient, contamination carried in by healthcare workers, visitors, etc, or residual contamination from prior occupants.³⁹ Many HAI-pathogens, such as, MRSA,VRE, C. difficile, and A. baumannii, can persist in a viable infectious state on environmental surfaces for days to months.^2,40

This study has some limitations. Our methods may underestimate total room MB and MDRO bioburden density in the rooms due to the variability of surface characteristics, microorganisms, and sampling efficiency. Additional sources of variablilty that we were unable to measure include the heterogeneity of surface contamination, and differing cleaning methods and cleaning intensities among facilities. Despite the increased labor in processing, we chose to use a large-surface-area sampling strategy to overcome the lack of surface homogeneity and to increase our sensitivity for detecting MDROs, especially in the terminal cleaned rooms. Although the relationships among sampled area, the distribution of sampled area across different sites, and sensitivity of MDRO detection are not fully known, sampling multiple sites did result in increased MDRO detection, as seen in the preliminary study (S1 and S2). Also, during the main study, collecting 2 composite samples increased not only the amount of bioburden recovered but also the percentage of MDRO-positive rooms compared to that expected if we had limited sampling to C1 or C2 sites (data not shown). In addition, we do not know what exact site the MDROs were recovered from due to the composite sampling strategy, and these results only provide a snapshot of room bioburden because the rooms were only sampled once. Another limitation is that we did not assess cleaning protocols or adherence to them in the rooms, so we cannot know whether these items/ rooms were cleaned properly or thoroughly. However, with the large number of rooms sampled, the effect of a few badly cleaned rooms would be minimized on the overall mean MB.

It will be important to determine how these levels of microbial contamination relate to the risk of the patient acquiring an MDRO. These data provide a first step in determining the MB of common hospital surfaces, which may help to develop standards for adequacy of cleaning and disinfection methods on healthcare surfaces and provides a framework for studies using the evidentiary hierarchy for environmental infection control to increase patient safety by cleaning and disinfection.³⁹ These results could be used to help parameterize models describing the role of environmental surface contamination in transmission. Future areas of investigation include the significance of cleaning type, cleaning products, and other variables on MB and risk of MDRO recovery. In addition, future studies should evaluate whether other sampling methods that are less labor intensive yield comparable bioburden or MDRO recovery as the sponge-wipe method.