Efficiency of four sampling methods used to detect two common nosocomial pathogens on textiles

Abstract

Detecting microorganisms on textiles is useful for many purposes, for example to determine the bioburden before laundering, assess the reduction in bacterial counts in connection with various laundry processes, or trace transfer routes in infection control investigations. Therefore a validated, reproducible and rational method is needed. For sampling microorganisms on textile surfaces the most commonly used method is the contact plate method using RODAC plates, first described by Hall and Hartnett followed by the swab sampling technique. Both methods can only capture microorganisms on the surface of the textiles while microorganisms that have penetrated into the deeper structure of the material will not be detected. In our research the contact plate method and the swabbing technique were compared with two wash-off methods. For the first wash-off method the destructive elution method was used, where microorganisms were eluted from the fabrics by shaking the fabrics for a certain time in an elution medium. For the fourth sampling method a nondestructive method that included a compact test device called Morapex® was used, which is based on forced desorption by pressing the microorganisms through the fabric without destroying the fabric.

In our research, two types of microorganisms were included (Klebsiella pneumoniae and Staphylococcus aureus) that cause common nosocomial infections. The aim of this study was to compare the efficiency of the four sampling methods for detecting microorganisms on textiles and to determine the lowest concentration, which can still be detected. The percentage of microorganisms that were detected by both elution methods was substantially higher than by sampling of fabrics with the contact plate method or swabbing. It can be concluded that a nondestructive method using a modified Morapex® device can be applied for quick determination of the hygienic condition of textiles.

Keywords

Textile hygiene swabbing RODAC plates elution method Morapex

Introduction

Detecting microorganisms on textiles is useful for many purposes, for example to determine the bioburden before laundering procedures, assessing the reduction of bacterial counts in connection with various laundry procedures, or trace transfer routes in infection control investigations.¹ The latter is especially important for hospital textiles² as they are a possible vehicle of nosocomial infections. The report written on the basis of recent national nosocomial infection prevalence surveys in Europe and based on the results of hospital-wide surveillance programmes of nosocomial bacteraemia in different European countries shows that the total patients acquiring a nosocomial infection in the EU every year can be estimated at 3,000,000. Approximately 50,000 deaths are estimated to occur every year as the consequence of the infection. Approximately 20% to 30% of nosocomial infections are considered to be preventable by intensive infection prevention and control programs including surveillance.^3,4 One of these surveillance programs is controlling the hygiene of hospital textiles.

The origin of infection can be an infected person or the environment. Microorganisms can be part of the patient's normal flora, which during the process of diagnosis, treatment and care of immunocompromised patients can cause the nosocomial infection. The source may also be other patients or health workers who are infectious. Microorganisms are able to survive on environmental surfaces for periods of up to several weeks,⁵ providing a significant biotransfer/cross-contamination/cross-infection potential⁶ that should not be overlooked. Microorganisms can also survive in the patient's abiotic environment, such as contaminated equipment for care, diagnosis and treatment (textiles), food, water, disinfectants and on surfaces,⁷ which shows that one of the possible sources of nosocomial pathogens can be inappropriately disinfected textiles.² Surveys show that hospital textiles can be the source of nosocomial infections with streptococci,⁸ enterococci,⁹ Bacillus cereus,¹⁰ staphylococci¹¹ and coliform bacteria.¹²

Hospital laundry needs to be ‘hygienically clean’, that is free of pathogenic microorganism in numbers sufficient to cause human illness.¹³ The concentrations or the infectious dose for pathogen bacteria can be from 1 to 100 units, where the immune status of the individual plays an important role;¹⁴ therefore the low detection limit of bacteria is very important.

In the recommendations of Centers for Disease Control and Prevention and the Healthcare Infection Control there is a report about the absence of microbiologic standards for laundered textiles, so no rationale exists for routine microbiologic sampling of cleaned healthcare textiles and fabrics. Sampling may be used as part of an outbreak investigation if epidemiologic evidence suggests that textiles, fabrics, or clothing are a suspected vehicle for disease transmission. Sampling techniques include aseptically macerating the fabric into pieces and adding these to broth media or using RODAC (replicate organism detection and counting) plates for direct surface sampling.¹⁵ In the European Union, as an important recommendation for hospital laundries, serves a valid Certificate of quality and hygiene in laundry,¹⁶ published by the Research Institute Hohenstein, Germany and authorized by the German Institute for quality assurance and certification (RAL). It follows the Robert Koch Institute requirements that indicate textiles as one of the critical control points where the sampling with RODAC agar plates should be conducted.¹⁷

Nosocomial infections do not only represent complications in the treatment of patients in the hospital, but also cause economic damage. A ten year survey between years 1990 and 2000¹⁸ shows that the total cost of nosocomial infections in developed countries has been estimated at 1 billion US$ in Europe, 5 to 10 billion US$ in the United States, for a total estimated 2 million cases per year. The overall additional costs associated with hospital-acquired infections in the ICU varied from $3000 to $40,000 per patient and are associated with an additional length of stay from 5 days to roughly 3 weeks. The mean cost of antibiotics has been estimated from $1000 to $16,000.¹⁸

Until now, two basic types of textile sampling methods, which vary depending on the state fabric at the end of the process, have been described:

Nondestructive methods, in which the test fabric remains essentially unaltered after sampling:

impression sampling, specifically RODAC or contact plating,¹⁹

release of fabric-bound dust and cellular particles onto sterile sampling surfaces by scraping with an inverted petri dish (sweep plating),²⁰

sampling by means of nutrient agar sausages,²¹

sampling by impact upon the fabric surface (percussion sampling),²²

the use of a Folin bubbler apparatus with attached funnel.^23,24

Destructive methods, in which the test fabric is rendered unsuitable for use after completion of the sampling process:

maceration^23,25,26 of fabric samples in a defined liquid medium,

agitation^13,27^–²⁹ of fabric samples in a defined liquid medium,

overlaying fabric samples with agar.^27,30

The German company, SedoTreepoint GmbH (http://www.sedo-treepoint.com/), reports about some typical and already used applications with the device called Morapex® for testing fabric (woven, nonwoven, knitted, yarn or fiber), paper and leather materials on a nondestructive basis. Typical Morapex® applications are pH measurement, control of wash procedure, wash and water fastness checks, perspiration fastness checks, residual analysis (including size, alkali, acids, salts, peroxide, formaldehyde, etc.) and conductivity analysis. This device has not been used for the elution or wash-out of microorganisms from fabrics.

This paper investigates the efficiency of four different sampling methods (swabbing, RODAC plates, classical elution method and the elution method using Morapex® device) for detecting two nosocomial pathogens S. aureus and K. pneumoniae (G+ and G- representative) on textiles. These microorganisms were chosen based on the results of research that indicate persistence on dry inanimate surfaces for up to seven months for S. aureus and even up to thirty months for K. pneumonia.³¹ The World Health Organization Publication also states these two species as one of the most common nosocomial pathogens among pathogenic bacteria.³² Based on our knowledge and literature review, we introduced a Morapex® A shown in Figure 1 for detecting microorganisms on textiles by elution for the first time.

Figure 1.

Device Morapex® A.

Materials and methods

Fabrics

100% cotton tabby weave fabric (thread spacing warp/weft 27 threads/cm; weight 190 g/m²), that is frequently used for hospital textiles such as pillow cases, bed sheets, quilt covers, etc., was used. Square pieces of 7 × 7 cm were cut. The pieces were sterilized in an autoclave at 121°C for 15 min and then dried in an oven at 100°C for 120 min. In a dust-free-chamber with UV light (UV MINI-V/PCR Telstar), the fabrics pieces were then transferred with sterile forceps to labelled petri dishes.

Microorganisms

Ready-made cultures of S. aureus and K. pneumonia were taken from the freezer and grown in nutrient broth (Tryptiyc Soy broth) for 4 days in an incubator at 37°C. Before each experiment viable counts of the culture were made, to enable the calculation of the number of cells inoculated onto the surfaces. Serial tenfold dilutions and viable plate counting using appropriate agars noted below for each microorganism were used. Work was carried out in a dust-free chamber with UV light (UV MINI-V/PCR Telstar).

Agars

Selective agars were used as a medium for incubating the microorganisms after retrieving from fabrics with different methods.³³ For S. aureus the Baird-Parker-agar base (incubation 48 hours at 37°C) with added egg-yolk tellurite emulsion was used. For K. pneumoniae the HiCromeKlebsiella Selective Agar Base (incubation 48 hours at 37°C) was used.

Application of microorganisms on fabrics

On each piece of fabric in the petri dish 2 mL of a prepared suspension of microorganisms was applied. All work was conducted in the dust-free chamber with UV light (UV MINI-V/PCR Telstar).

Petri dishes with inoculated fabrics were left in the dust-free chamber with UV light (UV MINI-V/PCR Telstar) for 24 hours, to allow the applied suspension to dry. Petri dishes with prepared pieces of fabrics were then sealed, and stored in the refrigerator.

Swabbing

Cotton swabs were pre-treated by dipping them into a prepared NaCl and Tween 80 solution (0.9% NaCl + 0.2% Tween 80). The tested fabric was swabbed firmly and evenly with one side of the swab in a horizontal direction, and with the other side in a vertical direction back and forth (one stroke back and one stroke forward) to cover the entire area. The swab was transferred into 20 mL NaCl and Tween 80 solution, vortexed for 30 s, and then left for 10 min and revortexed for 30 s.^34,35 The resulting suspension was again serially diluted and plated on the chosen selective agars. Work was carried out in a dust-free-chamber with UV light (UV MINI-V/PCR Telstar). After incubation, the colonies were counted and the colony forming units (cfu) were calculated.

RODAC plate method

The RODAC plates were prepared with selective agar for each of the two microorganisms. The RODAC plate was pressed onto the piece of fabric with a suspension and held for 3 s, followed by closing and placing into the incubator.¹⁹ Work was carried out in a dust-free-chamber with UV light (UV MINI-V/PCR Telstar). After incubation, the colonies were counted and the cfu was calculated,⁶ for each RODAC plate.

Destructive elution method

A piece of fabric with the suspension of microorganisms was transferred to a centrifuge tube with 20 mL of prepared physiological saline for shake-out (0.9% NaCl + 0.2% Tween 80)³⁶ using sterile tweezers. The centrifuge tube was closed and shaken for 10 min at 300 rpm on a shaking machine (Heidloph vibramax 100).

Serial tenfold dilutions and viable plate counting using appropriate agars for each microorganism were used to determine the cfu.⁶ Work was carried out in a dust-free chamber with UV light (UV MINI-V/PCR Telstar).

Nondestructive elution method using the orapex® device

The tested inoculated fabric was placed between two plates. Testing was conducted at room temperature. 20 mL test liquid (0.9% NaCl + 0.2% Tween 80) was pressed through the fabric in three cycles of 10 s. The extract was collected in a tube and again serial tenfold dilutions were prepared and viable plate counting using appropriate agars for each microorganism were used. After incubation, colonies were counted and the cfu was calculated.⁶

Results and discussion

The efficiency of swabbing is reliant on the efficiency of the individual carrying out three aspects of the procedure: the removal of bacteria from the surface; the removal of bacteria from the swab, and cultivation of bacteria.³⁷ In addition, the properties of the surface (topography, wettability, porosity, etc.) can affect the efficiency of swabbing,⁶ which is also the case when taking samples from textiles. The latter is also reflected in our survey as the lowest results of microorganisms retrieved from fabrics were achieved by swabbing. For both S. aureus and K. pneumoniae it was found that the cfu captured from inoculated fabric (with swabbing) was approximately 10⁵ cfu/mL lower than the initial applied concentration of microorganisms. Although swabbing is a widely used sampling method, it lacks the standardization required to provide the level of reproducibility and, as our research shows, it also gives the lowest results among the methods tested in our survey for fabrics. The lowest initial concentration of bacteria still detectable by this method was 5.0 × 10⁶ cfu/mL for S. aureus and 5.0 × 10⁷ cfu/mL for K. pneumoniae. When the initial bacterial concentration of K. pneumoniae inoculated onto the fabric swab was 5.0 × 10⁶ cfu/mL, no microorganisms were captured by the swabbing method. Lusardi et al.³⁸ carried out a laboratory investigation and validation of methods for sampling contaminated uniforms and work-wear; they report that the swabbing method gave low and inconsistent recoveries, which is probably because swabs are generally designed to access surface bacteria on wounds or work surfaces rather than reach contamination within the fibers of a material. Also the sampling head has a small surface area and may become overloaded. Alternative or supplementary methods are plentiful, but all have their limitations.⁶

The surface sampling method using RODAC contact plates filled with appropriate agars is one of the common used surface (also textiles) sampling methods originally described by Hall and Hartnett¹⁹ for use in direct contact sampling for surface contamination. The RODAC plate method has found wide acceptance and use in a variety of areas where sanitation and contamination level are important, particularly in institutional areas, such as hospitals and food production and service facilities. The use of RODAC plates for surface sampling can be valuable in the field because of its simplicity, reliability, and transportability, particularly in estimating the bacterial contamination on flat surfaces.³⁹ The efficiency of this method depends on the evenness of the surface tested⁴⁰ and because the structure of a fabric, due to the mechanical combination of warp and weft, a relatively small contact area is available. This is clearly seen in results from our survey, where the cfu captured from the inoculated fabric (with RODAC plates) were also approximately for 10⁵ cfu/mL lower from initial applied concentration of microorganisms. The low counts of microorganisms covered by the RODAC plate technique appear in several studies.⁴¹ Eriksson et al.⁴² report much lower counts from impression plates (RODAC agar plates) than those from the elution method and therefore conclude that the contact plate technique in inappropriate for determination of bioburden on textiles. The other problem obtained with our work was that when sampling fabrics with higher initial concentration of microorganisms, counting the cfu on RODAC plates was very difficult or even sometimes impossible. This is why contact plates are more successful if a selective culture media is used for particular indicator microorganisms on a surface, as suggested by Egington et al. in 1995.³² If surfaces are rough, or wet, then the sampling is inaccurate, or the resulting growth on the agar may be confluent.³² The usefulness of RODAC plates can be limited to surfaces with lower microorganism concentrations or for verifying whether a microorganism is present or not at all.

Much better results were obtained when using both the destructive and the nondestructive wash-out or elution methods, where the cfu captured from the inoculated fabrics (with elution) were approximately 10³ cfu/mL lower than the initial applied concentration of microorganisms. According to Cody et al.,⁴³ the elution method has excellent overall recovery efficiency, is easily performed, and yields reproducible results under a variety of test conditions, bacterial species, seed concentrations and fabric types. Our results also confirm these conclusions. The destructive elution method, although providing recovery data comparable to that of the maceration method, is simpler, quicker, and less expensive to use. Cody et al.³⁸ suggests that the elution method can be used as a simple and accurate method for enumerating fabric-associated bacteria and will permit assessment of bactericidal characteristics of laundry procedures. The downside of this method is that it belongs to a group of destructive methods, where a piece of fabric must be cut out of the sampling object, which means that the test fabric is rendered unsuitable for use after completion of the sampling process. So this method can be particularly useful in the laboratory criteria, but it cannot be used in a real environment.

The Morapex® device is able to analyse and therefore control either raw or dyed material in minutes rather than hours, compared to standard methods. Morapex® A is a compact test device for use in production and can operate up to 95°C; the multi liquor option is able to operate with a wide range of wash liquids. When using the Morapex device for eluting microorganisms from inoculated fabrics, the efficiency was similar to the classical elution method. Cfu captured from inoculated fabric (with Morapex) were approximately 10³ cfu/mL lower than the initial applied concentration of microorganisms. The Wilcoxon Signed Rank Test does not show a statistical significance of the difference (S.aureus: p = 0.116; K.pneumoniae: p = 0.173) between sampling with the Morapex device and the destructive elution method about which Cody et al.³⁸ reports yielded reproducible results under a variety of test conditions, bacterial species, seed concentrations, and fabric types. The system worked according to the method of forced desorption, which means the inner condition of a fabric was revealed. The testing material was placed between two metal plates; the test liquid was pressed through the fabric and then collected in a tube. Such testing is possible at any stage of production, for example, on incoming fabrics, intermediate analysis during production, analysis of finished goods, research and development checks, the effect of process and equipment adjustments, etc. In our research, the device has also been tested for the detection of microorganisms, therefore it can also be used as an efficient and nondestructive method for checking the hygiene of textiles, which is an important aspect in preventing nosocomial infections. The obtained extract can be analysed quickly and easily and the tested fabric remains essentially unaltered after sampling and can therefore be reused in the real environment.

Precise recovery rates of microorganisms for different initial applied concentrations are graphically presented in Figure 2 and Figure 3. When sampling S. aureus at the initial applied concentration 5 × 10⁸ cfu/mL, the results for sampling with different techniques were 9.13 × 10³ cfu/mL for swabbing, 5.50 × 10³ cfu/mL for RODAC agar plates, 3.56 × 10⁶ cfu/mL for destructive elution method and 4.88 × 10⁵ cfu/mL for Morapex. At the initial concentration of 5 × 10⁷ cfu/mL, the rates of obtained microorganisms were 4.97 × 10² cfu/mL for swabbing, 3.41 × 10³ cfu/mL for RODAC agar plates, 4.23 × 10⁵ cfu/mL for destructive elution method and 1.78 × 10⁵ cfu/mL for Morapex. At the lowest (5 × 10⁶ cfu/mL) initial applied concentration of microorganisms, the results were 4.72 × 10 cfu/mL for swabbing, 8.53 × 10 cfu/mL for RODAC agar plates, 6.32 × 10³ cfu/mL for the destructive elution method and 8.30 × 10³ cfu/mL for Morapex.

Figure 2.

Sampling Staphylococcus aureus with various methods. Initial bacterial concentration on fabric pieces before overnight drying: (A) 5 × 10⁸ cfu/mL, (B) 5 × 10⁷ cfu/mL, (C) 5 × 10⁶ cfu/mL.

Figure 3.

Sampling Klebsiella pneumoniae with various methods. Initial bacterial concentration on fabric pieces before overnight drying: (A) 5 × 10⁸ cfu/mL, (B) 5 × 10⁷ cfu/mL, (C) 5 × 10⁶ cfu/mL.

When sampling K. pneumonia at the initial applied concentration of 5 × 10⁸ cfu/mL, the results for sampling with different techniques were 3.17 × 10³ cfu/mL for swabbing, 3.93 × 10³ cfu/mL for RODAC agar plates, 3.39 × 10⁵ cfu/mL for the destructive elution method and 1.07 × 10⁶ cfu/mL for Morapex. At the initial concentration of 5 × 10⁷ cfu/mL, the rates of obtained microorganisms were 5.45 × 10² cfu/mL for swabbing, 2.44 × 10³ cfu/mL for RODAC agar plates, 8.72 × 10⁴ cfu/mL for the destructive elution method and 5.38 × 10⁴ cfu/mL for Morapex. At the lowest (5 × 10⁶ cfu/mL) initial applied concentration of microorganisms, there were no microorganisms retrieved with swabbing, 1.66 × 10² cfu/mL for RODAC agar plates, 4.25 × 10³ cfu/mL for the destructive elution method and 1.12 × 10⁴ cfu/mL for Morapex.

The main difference between the four types of sampling methods mentioned above is that microorganisms that have penetrated into the deeper structure of the material will not be detected by the RODAC plate method,¹ neither by swabbing, but they can be captured by elution, either destructive or nondestructive. The accuracy of sampling with swabbing or RODAC agar plates relies on the efficiency of the individual carrying out the procedure, which is not the case in both elution methods where the process is automated and the elution conditions are constant.

Conclusion

Most methods for sampling microorganisms on textiles have certain limitations. The downside of the swabbing and RODAC plate method is the low efficiency of capturing microorganisms due to the rough, uneven three-dimensional fabric surface. The most applicable methods are the methods that work on the principle of eluting microorganisms from textiles as microorganisms in the fabric are also collected. The Morapex® A device has proved to be a better implementation for textile hygiene testing in a real environment as it is a nondestructive elution based method.

Further work will be focused on PCR (polymerase chain reaction) detection of microorganisms in samples collected with the elution method. Implementation of classical methods takes between 2 to 4 days followed by phenotypic identification of the microorganisms present, while PCR detection of microorganisms can be completed within a few hours. This molecular based method will enable faster, more reliable and efficient detection of nosocomial pathogens on textiles.

Footnotes

Acknowledgements

We are very grateful to SedoTreepoint GmbH, Germany, for the rental of the Morapex® A device.

Funding

This work was supported by the ARRS 1000-10-310152.

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