Evaluation of environmental sampling methods for detection of Salmonella enterica in a large animal veterinary hospital

Abstract

Environmental surveillance for Salmonella enterica can be used for early detection of contamination; thus routine sampling is an integral component of infection control programs in hospital environments. At the Purdue University Veterinary Teaching Hospital (PUVTH), the technique regularly employed in the large animal hospital for sample collection uses sterile gauze sponges for environmental sampling, which has proven labor-intensive and time-consuming. Alternative sampling methods use Swiffer brand electrostatic wipes for environmental sample collection, which are reportedly effective and efficient. It was hypothesized that use of Swiffer wipes for sample collection would be more efficient and less costly than the use of gauze sponges. A head-to-head comparison between the 2 sampling methods was conducted in the PUVTH large animal hospital and relative agreement, cost-effectiveness, and sampling efficiency were compared. There was fair agreement in culture results between the 2 sampling methods, but Swiffer wipes required less time and less physical effort to collect samples and were more cost-effective.

Introduction

Risk of infection is increased in hospitalized animals because of the stress of transportation and hospitalization and the close proximity to other animals that are likely to be shedding infectious agents (1,2). Creating a protective environment for hospitalized animals, clients, and hospital personnel is part of the ethical and professional responsibility of veterinarians, who are obliged to minimize infectious disease hazards in veterinary hospitals (3,4). Animals in the community, such as on the farm, and others which may have contact with patients after discharge from hospital, are also at risk for disease transmission and therefore represent a further ethical obligation for veterinarians in terms of preventing the spread of disease outside the hospital environment (4). Thus, coordinated infection control practices are necessary for optimization of patient care at veterinary hospitals and disease control within and outside the confines of the hospital environment. These control measures should be used routinely and should be periodically evaluated in order to ensure that occurrences of hospital-acquired disease are minimized (5,6).

Salmonellosis has historically been the most prevalent hospital-acquired infection (HAI) in veterinary teaching hospitals (1,7) and outbreaks of Salmonella spp. infections have resulted in hospital closures leading to significant financial losses as well as losses in client confidence, employee morale, and student educational opportunities (1). In one outbreak, financial losses resulting from hospital closure were estimated at $4.12 million (USD) and students were required to go out of state to complete their large animal clinical training (8). Also, in addition to the impact HAIs have on patients, a survey of 38 veterinary hospitals found that hospital personnel at 50% of hospitals had reported significant health problems attributable to Salmonella enterica infections in the 2 y before the interview (7), highlighting the risk of zoonotic disease transmission in veterinary hospitals. This is especially problematic given that data suggest HAIs can occur for a number of months before patterns in clinical disease are recognized (1); yet multiple veterinary hospitals have reported that surveillance activity is only initiated when there is a perceived increase in HAIs rather than being conducted routinely (7). This highlights the critical need for the development and utilization of sensitive sampling and culture systems (4) for early detection of infectious disease hazards.

Environmental surveillance for Salmonella species can be considered an integral part of a routine surveillance protocol (9,10) and has been shown to aid in the mitigation of at least 2 outbreaks in large animal veterinary teaching hospitals (8,11). Current Salmonella sampling methods used in large animal veterinary teaching hospitals in the US are not standardized (7) and there is no consensus regarding which detection method is best (4,9,12). The absence of standardized surveillance techniques has led to a corresponding lack of objective assessments of specific infection control practices, especially in relation to implementation within the veterinary hospital environment (4).

To date there has only been 1 hospital-based study directly comparing 2 of these methods: gauze sponges and electrostatic (Swiffer) wipes (9). In that analysis, the use of electrostatic wipe sampling and culture method was found to be more sensitive than the sponge method and samples were considered easier to collect using the electrostatic wipes (9). The protocol for the environmental sampling method used in the large animal hospital at the Purdue University Veterinary Teaching Hospital (PUVTH) was established in 2000 in response to an outbreak of salmonellosis (13). Under the current protocol, detection of Salmonella spp. in the environment results in extensive cleaning and disinfection in the areas of the hospital in which Salmonella spp. was detected (14). Upon contemporary evaluation of the environmental sampling protocol using guidelines established by the US Center for Disease Control and Prevention (CDC) (6), it was decided that the monitoring system lacked flexibility and timeliness, attributes considered necessary for optimal surveillance systems. Therefore, the purpose of this study was to directly compare the current environmental sampling and culture methods to the electrostatic wipe sampling and culture method in order to determine which surveillance system would be the most useful for detection of S. enterica in the large animal hospital at PUVTH.

Materials and methods

Study overview

Environmental samples were collected using 2 previously published environmental sampling methods (9,13) for detection of S. enterica in the large animal hospital at the PUVTH in June 2015. Relative agreement between culture results obtained using both methods, cost-effectiveness, and sampling efficiency of the 2 methods were compared.

Surveillance and infection control methods

As part of the current surveillance methods employed by the Infection Control Committee (ICC) at PUVTH, environmental samples are routinely collected from the large animal hospital biannually (once in May/June and once in September/October). These samples are collected from floor drains, walls, hard surfaces that are touched by hands and floors in areas that have been cleaned using a standardized protocol including not more than 24 h before sample collection (14). Briefly, cleaning protocols include removal of debris, application of 10% bleach and soap as needed, and weekly disinfection with a quaternary ammonia compound. Drains are flushed with water and approximately 7.6 L (2 gallons) of disinfectant once per week and stalls are scrubbed with bleach and soap followed by disinfection after discharge of hospitalized patients.

Environmental sampling

Two sample collection devices were used for collection of environmental samples in this study: individually packaged gauze sponges and electrostatic (Swiffer) wipes. Established protocols were used for both methods (9,13), which resulted in several differences between the 2 sampling procedures. In the method established by the ICC in 2000, gauze sponges were moistened with sterile saline and held with forceps, which were disinfected with 70% ethanol between samples. Sterile gloves were also worn, and a new set of gloves was used for each sample. Sponges were individually placed in non-sterile plastic bags and transported to the laboratory immediately following sampling each day. For the electrostatic wipe method, wipes were attached to a Swiffer sweeper mop using non-sterile gloved hands during sampling of walls and floors and were held in a non-sterile gloved hand for collection of samples in small spaces. New non-sterile gloves were worn for each sample collected. After sampling, wipes were placed into pre-labeled sterile Whirl-Pak bags and immediately transported to the laboratory. In between samples, the sweeper mop was disinfected with 70% ethanol and allowed to dry. An approximately equal surface area was sampled using each of the collection methods.

Sampling by both methods was conducted in parallel within a period of 2 wk in June 2015. A total of 245 individual samples were collected using the gauze sponge method, the sampling protocol used by the PUVTH ICC. Electrostatic wipes were used to collect a total of 50 samples in the same areas of the hospital and at the same time as those sampled using the gauze sponge method. Though the same environments were sampled with each method, fewer individual samples were taken using the Swiffer method as many of the samples were consolidated. For example, banks of stalls in each ward were combined as opposed to individually sampling each stall. This was done in an effort to facilitate more routine sampling in the PUVTH large animal hospital by reducing the total number of samples taken during each sampling period.

Culture procedures

All bacterial cultures were carried out at the Purdue Animal Disease Diagnostic Laboratory (ADDL). Samples collected using gauze sponges were placed in 100 mL of tetrathionate broth and incubated for 48 h at 37°C. They were then plated on xylose lysine tergitol-4 (XLT-4) agar and incubated for another 48 h at 37°C, then plated on brilliant green agar and incubated for 24 h at 37°C. Samples collected using electrostatic wipes were placed in 90 mL of buffered peptone water and incubated for 24 h at 42°C. Following this, 1 mL of the buffered peptone water was transferred to 10 mL of tetrathionate broth with iodine and incubated for 24 h at 42°C, then 100 μL of the tetrathionate broth culture was added to 10 mL of Rappaport-Vassiliadis R10 broth and incubated for 24 h at 42°C. Each sample was then plated on XLT-4 agar and incubated for 48 h at 35°C. Colonies suspected to be S. enterica based upon appearance were identified using the matrix-assisted laser desorption ionization — time of flight mass spectrometry [(MALDI-TOF MS) Biotyper; Bruker, Billerica, Massachusetts, USA], and isolates were submitted to the National Veterinary Services Laboratory (NVSL) for serotyping.

Financial analysis

Direct costs for each sampling method were calculated by adding the prices of all the equipment and sampling materials; they were then compared. The number of person-hours needed to collect samples using each method were also compared on an hour-per-hour basis.

Data analysis

Culture results were described using descriptive statistics. Cohen’s κ was used to evaluate agreement between the culture results collected with gauze sponge and electrostatic wipes using the following scale: 0 = no agreement; 0.01 to 0.20 = slight agreement; 0.21 to 0.40 = fair agreement; 0.41 to 0.60 = moderate agreement; 0.61 to 0.80 = substantial agreement; and 0.81 to 1.0 = near perfect agreement. All analyses were performed using Stata SE13.1 (Stata, College Station, Texas, USA).

Results

Salmonella enterica was cultured from 4% (2/50) of samples collected using electrostatic wipes and 1.2% (3/245) of samples collected using gauze sponges (Table 1). Salmonella enterica was cultured from 2 floor drains and the exterior of 1 water hose in different areas of the hospital using gauze sponges, and from floor surfaces in 2 different areas of the hospital using electrostatic wipes (Figure 1). In only 1 area of the hospital were positive samples detected using both collection methods. The κ statistic (κ = 0.37) indicated fair agreement between the 2 methods in terms of ability to detect S. enterica from the environment, though the low prevalence of positive culture results may have affected this value.

Table 1.

Culture results from environmental samples obtained using the gauze sponge and electrostatic wipe methods; sample pairs based upon area of the hospital in which the sample was collected.

Gauze sponge sampling	Electrostatic wipe sampling
	Negative	Positivea	Total
Negative	46	1	47
Positiveb	2	1	3
Total	48	2	50

^aPositive samples were detected from 2 floor surfaces in the large animal hospital, 1 of which was in the same location from which a positive sample was collected from a floor drain using a gauze sponge.

^bPositive samples were detected from the inside of 2 floor drains in different locations within the large animal hospital and from the exterior surface of a water hose.

Figure 1.

Map of the large animal hospital at PUVTH. Locations of electrostatic wipe samples (Circles) and gauze sponge samples (Diamonds) which cultured positive are shown.

Financial analysis showed that the total cost of supplies for the gauze sponge method was $278.10, while the total cost for the electrostatic wipe method was $263.68 (Table 2); however, most of the expenses for the electrostatic wipe method were non-recurring fees, such as for the sweeper mops, ethanol, spray bottles, and caddy. Fees for bacterial culture were $3715 for the gauze sponge samples and $760 for the electrostatic wipe samples. Time spent was estimated to be between 16 to 40 person-hours for the gauze sponge method, and 1 to 2 person-hours for the electrostatic wipe method. It was also noted that use of the Swiffer sweeper mops facilitated sample collection over large surfaces such as walls and floors.

Table 2.

Direct costs for the gauze sponge and electrostatic wipe sampling methods used in the same areas of the large animal hospital at PUVTH.

Gauze sponge protocol (245 samples) ($)		Electrostatic wipe protocol (50 samples) ($)
Sterile prep basins	20.00	Sweeper mops	55.96
Sterile gloves	200.00	Swiffer wipes	49.65
4 × 4 trays	12.00	Whirl-Pak bags	76.56
Lactated Ringer’s	9.25	Examination gloves	37.00
Sponge forceps	12.00	Ethanol	38.03
Sample bags	6.25	Spray bottle	1.76
Accela	18.60	Caddy	4.72
Subtotal	278.10	Subtotal	263.68
Laboratory fees	3715.00	Laboratory fees	760.00
Total	$3993.10	Total	$1023.68

^aHydrogen peroxide cleaner and disinfectant.

Discussion

This study suggests that culture results obtained from environmental samples collected using either the gauze sponge or electrostatic wipe technique are similar; however, use of electrostatic wipes required less time and, subjectively, less physical effort to collect samples. Furthermore, the electrostatic wipe sampling protocol was more cost-effective than the gauze sponge protocol. Because electrostatic wipes are less expensive and more efficient in terms of time and physical effort, their use could facilitate determination of the baseline rate of positive S. enterica cultures within a veterinary hospital setting. Records from PUVTH show that in the past, environmental samples were collected using gauze sponges only biannually because of their high cost and the amount of physical effort required for their collection, resulting in insufficient data to determine the baseline rate of contamination within the hospital or to allow for evaluation of trends. Therefore, in the PUVTH large animal hospital, use of electrostatic wipes for environmental sample collection is preferable to use of gauze sponges for routine environmental surveillance.

This study was limited by the fact that not all samples taken with gauze sponges were directly matched with samples taken with electrostatic wipes. Since some areas of the hospital were only sampled with sponges the figures for total cost and physical effort may be slightly underestimated for electrostatic wipes. However, in areas that were sampled with both methods, each method was equally able to detect the presence of S. enterica. Also, the sponges were used to sample inside floor drains, 2 of which cultured positive for S. enterica. In contrast, electrostatic wipes were only used to sample the floor surfaces, including the tops of each drain but not the inside the drain. The clinical significance of finding S. enterica at the bottom of a drain is unknown, but in this case only 1 location corresponding to the drains that cultured positive using gauze was found to be culture positive using the electrostatic wipes. In addition, the reliability of the Kappa statistic used may have been negatively affected by the low prevalence of positive samples. Finally, it is important to note that the use of enrichment in the culturing procedure makes the results solely qualitative; therefore, analysis of surveillance data over time will consider positive versus negative cultures only, and not level of contamination (9,10).

There has been only 1 study directly comparing the sensitivity of pre-moistened sponges with electrostatic wipes in a veterinary hospital environment (9). In this study, the wipes were found to be 3 times more sensitive in detecting the presence of Salmonella, which was recovered from 14% of the electrostatic wipe samples but only 4% of the sponge samples (9). This difference in sensitivity could be due to either a difference in ability to collect microorganisms and/or differences in laboratory culture techniques (9,14). Likewise, in the current study, samples collected with electrostatic wipes appeared to be more likely to detect the presence of Salmonella compared with samples collected using gauze sponges. The reason for this apparent difference in sensitivity could be either the collection or culture techniques used.

At the University of Pennsylvania, investigators found that the ability to detect environmental Salmonella appeared to be vastly improved after switching from gauze sponges (8.8% positive) to electrostatic wipes (30% positive) during an active outbreak, and that the electrostatic charge of the wipes combined with the ability to sample larger areas led to more frequent recovery of bacteria (8). This change in protocol helped to reveal that Salmonella was present in areas of the hospital that had previously been culture-negative, but where animals were continuing to acquire Salmonella infections. Use of gauze sponges resulted in the false assumption that the hospital was not contaminated with Salmonella, but modifying the surveillance techniques to include electrostatic wipes revealed that the sponges failed to detect extensive environmental contamination. This shows that use of electrostatic wipes could potentially increase the efficiency of environmental sampling used to detect Salmonella in large animal hospitals, which could create a more robust routine surveillance system and optimize patient care.

Electrostatic wipes were also shown to be useful as an aid to mitigation during a Salmonella outbreak at Colorado State University (CSU) in 2006 (11). Active environmental surveillance using electrostatic wipes enabled early detection of the strain of Salmonella that was causing the outbreak within the hospital environment. Increased cleaning and disinfection of the hospital environment was initiated after the environmental contamination was detected. This prevented further transmission of Salmonella and hospital closure was avoided (11) and contrasts with what occurred during a similar outbreak at CSU in 1996. During the 1996 outbreak, positive environmental cultures were detected at a rate of 2.5% using gauze sponges; in 2006, the rate of positive cultures was 24.5% (days 1 to 35 of outbreak) using electrostatic wipes (11). In the wake of this most recent outbreak it was concluded that aggressive surveillance with electrostatic wipes contributed to avoidance of hospital closure (11). In 2000, PUVTH experienced an outbreak that resulted in hospital closure, during which approximately 1100 environmental samples were taken. Salmonella was isolated from only 1.1% of the samples; most of those positive isolates were detected from drains located inside patient stalls (13). Use of electrostatic wipes would aid in early detection, facilitating early intervention in order to curb future outbreaks and prevent hospital closure.

Though standardization of environmental surveillance techniques across hospitals would facilitate research by providing reliability and comparability of data (4), infection control programs have to be customized to fit the needs of individual hospitals (15). There is insufficient research to indicate whether regularly conducted active surveillance as reported here is preferable to alternate strategies of infection control such as passive collection of diagnostic reports or test results. However, more visible efforts are inherently accompanied by increased awareness of infection control procedures (7) and detection of environmental contamination before an outbreak of disease occurs is also a valuable outcome (7). In conclusion, with the significant variability in Salmonella sampling and culture systems used in large animal veterinary teaching hospitals, more studies of the kind reported here are needed to allow for direct comparison and evaluation of the efficacy of these methods (7,9) and to help facilitate customization of infection control programs. CVJ