Real-time polymerase chain reaction for quantitative assessment of common pathogens associated with healthcare-acquired infections on hospital textiles

Abstract

A hospital environment may act as a significant reservoir for potential pathogens that can be transmitted with hospital textiles, which could represent a source of healthcare-acquired infections. Quantitative assessment of nosocomial pathogens with real time polymerase chain reaction (qPCR) on textiles can serve to verify the achievement of standards for textile hygiene of hospital laundry that assess the risk for acquiring hospital infection from inappropriately disinfected textiles. The aim of the study was to establish qPCR for quantitative assessment of selected common nosocomial pathogens (Clostridium difficile, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa) on hospital textiles and to compare the efficiency of the molecular method to the standard procedures for evaluating the bio burden of textiles in hospitals. This study demonstrated that presence of nosocomial pathogens on hospital textiles can be confirmed with qPCR even where conventional techniques do not give any results. qPCR offers a possibility to confirm the presence of microorganisms in dead or viable but non-culturable states that cannot be detected by conventional sampling techniques but may still pose a hazard to public health.

Keywords

healthcare-acquired infections hospital textiles

The hospital environment provides an important ecological niche for organisms that could have clinical significance^1,2 and may well act as a significant reservoir for potential pathogens,³ which can then be extended by vectors such as air turbulence, aerosolized moisture, an unwashed hand or direct contact with an inanimate object, equipment or material.^3,4 The majority of microorganisms that are most frequently identified as the cause of healthcare-acquired infections (HCAIs) are ubiquitous gram-negative or gram-positive bacteria, which can be isolated on a large variety of inert surfaces found in hospitals and on human skin, including the hands of medical staff.⁵ The environment can play a marked role in the nosocomial transmission of microorganisms,⁶ where garments of healthcare workers are an important aspect of the environment that can easily become contaminated. It has been suggested that hospital textiles could be a source of HCAIs, contributing to the transmission of pathogens both through indirect contact, via hospital staff, endogenously, and by means of aerosols.^6,7 The characteristics of the textile in question, together with humidity and heat, can create the right conditions for the proliferation of numerous microorganisms.^8,9 Boyce¹⁰ reported that 65% of nurses who had performed patient care activities on patients having MRSA in a wound or urine had contaminated their nursing uniforms or gowns with MRSA. Pathogenic bacteria such as P. aeruginosa and K. pneumoniae¹¹ and C. difficile¹² were also detected on uniforms of physicians and nurses.

The standard microbial procedure for evaluating the bio burden of textiles in hospitals is sampling with RODAC agar plates and identifying the pathogen by gram stain and biochemical tests, which will take about two to four days in a microbiological laboratory.¹³ The polymerase chain reaction (PCR) is a powerful technique that exponentially amplifies a small amount of deoxyribonucleic acid (DNA) into a large amount by repeating a simple three-step process: denaturation, annealing and synthesis. Real time PCR (qPCR) is a technique used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, complementary DNA (cDNA) or ribonucleic acid (RNA)) can be quantified.¹³ qPCR is based on the detection of the fluorescence produced by a reporter molecule, which increases as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. The fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green) or sequence-specific probes (i.e. Molecular Beacons or TaqMan® Probes). qPCR facilitates the monitoring of the reaction as it progresses.¹³ Quantitative assessment of nosocomial pathogens on textiles can serve to verify the achievement of standards for textile care of hospital laundry, such as RAL-GZ 992/2¹⁴ and the EN 14065 RABC standard.¹⁵ Such standards also assess the risk for acquiring infection in hospital.¹⁶

The present study introduces qPCR for quantitative assessment of common pathogens associated with HCAI on hospital textiles. Four of the most common nosocomial pathogens were included: Clostridium difficile, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa.

Materials and methods

Textile swatches

Sterile 100% cotton tabby weave fabric (thread spacing of warp and 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. Single-layer textile square pieces of 7 cm × 7 cm were cut from new, not used, textile. The pieces were sterilized in an autoclave at 121℃ for 15 min and then dried in an oven at 100℃ for 120 min. In a dust-free chamber with ultraviolet (UV) light (UV MINI-V/PCR Telstar), the fabrics pieces were then transferred with sterile forceps to labeled Petri dishes. To confirm sterility of textile swatches before inoculation, three pieces were treated with the elution method described below.

Challenge microorganisms

An overnight culture of the following challenge microorganisms was used: Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 13883) and Pseudomonas aeruginosa (ATCC 27853).

For Clostridium difficile (ATCC 9689), spore preparation a five-day-old culture grown on selective medium for C. difficile was swabbed and resuspended in 1 ml sterile distilled water. Spore suspension was stored at 4℃ until use in experiments.

Inoculation of textiles swatches

Previously sterilized textiles swatches were inoculated with 2 ml of a prepared suspension of microorganisms / spores in different concentrations and left in laminar flow for 24 hours, to allow the applied suspension to dry.

Determining the efficiency of autoclaving and soaking in sodium hypochlorite

To test the efficiency of autoclaving and soaking in sodium hypochlorite, textile swatches were inoculated with 2 ml of a prepared suspension of microorganisms/spores as follows: six pieces with a suspension of C. difficile spores (2.8 × 10⁷cfu/ml) and six pieces with a suspension of S. aureus (6.2 × 10⁷cfu/ml) and left overnight in laminar flow at room temperature to dry.

For each microorganism three pieces were autoclaved for 30 min and the other three pieces were soaked in 3% sodium hypochlorite for 10 min. The treated pieces were then treated with the elution method described below. The presence of microorganisms was tested by the viable plate counting method and the molecular methods in real time described below.

RODAC plate sampling technique

The RODAC plates were prepared with selective agars for each of the challenge microorganism (i.e. Clostridium Difficile Agar for C. difficile, Baird-Parker agar for S. aureus, HiChrome Klebsiella selective agar for K. pneumoniae and Cetrimide agar for P. aeruginosa). The appropriate RODAC plate was pressed onto the inoculated textile swatch and held for 3 seconds, followed by closing and placing into the incubator. After incubation, the colonies were counted and the cfu (colony forming unit) was calculated for each RODAC plate.

Elution method

For the elution method, an inoculated textile swatch was transferred to a centrifuge tube with 20 ml of prepared physiological saline for shake-out (0.9% NaCl + 0.2% Tween 80) (JIS L 1902¹⁷) using sterile tweezers. The centrifuge tube was closed and shaken for 10 min at 300 rpm on a shaking machine (Heidloph vibramax 100; Figure 1). Serial ten-fold dilutions and viable plate counting using appropriate agars for each microorganism were used to determine the cfu.

Figure 1.

Standard curves and correlation coefficients for all four challenged pathogens.

DNA extraction

Bacterial genomic DNA was extracted from the suspension of microorganisms retrieved from textiles with the elution method. Extraction was performed with PrepMan Ultra Sample Preparation Reagent (Applied Biosystems) for each sample in accordance with manufacturer's instructions. Extracted DNA was stored at −20℃ prior to PCR amplification. Bacterial genomic DNA extracted from an overnight culture in liquid broth was used as a positive control.

Oligonucleotide primer selection

The target genes for the four challenge microorganisms, their oligonucleotide primer pairs and their respective amplicon sizes are shown in Table 1.

Table 1.

Oligonucleotides used for polymerase chain reaction amplification

Target	Primer	Primer 5′——-3′	Size of product (bp)
Clostridium difficile	CD⁴⁰	f (5′-TTG AGC GAT TTA CTT CGG TAA AGA-3′)	157
Clostridium difficile	CD⁴⁰	r (5′- CCA TCC TGT ACT GGC TCA CCT-3′)
Staphylococcus aureus	egcAU⁴¹	f (5′- CTTCATATGTGTTAAGTCTTGCAGCTT-3′)	82
Staphylococcus aureus	egcAU⁴¹	r (5′-TTCACTCGCTTTATTCAATTGTTCTG-3′)
Klebsiella pneumoniae	ITS^a,42	f (5′-ATT TGA AGA GGT TGC AAA CGA T-3′)	130
Klebsiella pneumoniae	ITS^a,42	r (5′-TTC ACT CTG AAG TTT TCT TGT GTT C-3′)
Pseudomonas aeruginosa	gyrB ⁴³	f (5′-CCT GAC CAT CCG TCG CCA CAA C-3′)	222
Pseudomonas aeruginosa	gyrB ⁴³	r (5′-CGC AGC AGG ATG CCG ACG CC-3′)

16S–23 S rDNA internal transcribed spacer

Polymerase chain reaction amplification

qPCRs were performed in duplicates using a StepOne Real Time PCR System (Applied Biosystems). The optimized SYBR Green PCR reaction mixture contained the following: Power SYBR Green PCR Master Mix (Applied Biosystems); 300 nM of each primer for C. difficile, S. aureus and K. pneumoniae or 100 nM of each primer for P. aeruginosa; 1 µl of DNA and PCR water to a final volume of 10 µl. The amplification conditions for each microorganism are shown in Table 2. A melt curve was performed at the end of each PCR run. In each qPCR experiment, serial ten-fold dilutions of standard DNA and a negative control, both in duplicate, were included. After each run baseline was automatically set and threshold value Ct was determined.

Table 2.

Amplification and melt curve conditions

Step	Clostridium difficile	Staphylococcus aureus	Klebsiella pneumoniae	Pseudomonas aeruginosa
Initial heat activation	95℃ 10 min
Denaturation	94℃ 30 s	95℃ 15 s	94℃ 15 s	95℃ 3 s
Annealing	68℃ 30 s	61℃ 30 s	58℃ 15 s	60℃ 30 s
Extension	72℃ 30 s	72℃ 30 s	72℃ 15 s
Final extension	72℃ 10 min
Melt curve	95℃ 15 s	95℃ 15 s	95℃ 15 s	95℃ 15 s
	60℃ 1 min	40℃ 1 min	60℃ 1 min	60℃ 20 s
	↑ + 0.5℃	↑ + 1℃	↑ + 0.3℃	↑ + 0.2℃
	95℃ 15 s	95℃ 15 s	95℃ 15 s	95℃ 15 s

Quantification of real time polymerase chain reaction results

To create standard curves, six ten-fold serial dilutions of the DNA isolated from each challenge microorganism cell suspension in concentration 1.7 × 10⁸cfu/ml for C. difficile, 6.8 × 10⁷cfu/ml for S. aureus, 4.5 × 10⁹cfu/ml for K. pneumoniae and 7.1 × 10⁷cfu/ml for P. aeruginosa were used. After amplification results were plotted against the quantity of gene copies, linear regression was used to calculate the slope (S) of the standard curves and correlation coefficient (R²) for each experiment (Figure 1). Using these standard curves, from Ct values determined for each sample the cell equivalent values (cevs) were determined. The experiment for each challenged pathogen was repeated three times and mean values were calculated.

Detection of amplicons

Following amplification, aliquots (3 µl) were removed from each reaction mixture and examined by electrophoresis (100 V, 60 min) in gels composed of 1.5% (v/v) agarose (Sigma) in 0.5 Tris/Borate/ethylenediaminetetraacetic acid (TBE) buffer (89 mM Tris base, 89 mM Boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA)) stained with SYBR Green I nucleic acid gel stain (Sigma Aldrich). Gels were visualized under UV illuminator Transiluminator Super-Bright (VilberLourmat) at 312 nm using a gel images system Doc Print VX2 (VilberLourmat) to confirm the presence of the amplified DNA. Images were transferred to a PC and processed by the Photo-Capt software.

Results and discussion

Efficiency of autoclaving and soaking in sodium hypochlorite

The efficiency of autoclaving textile swatches inoculated with C. difficile or S. aureus, soaking textile swatches in sodium hypochlorite solution and confirmation of fabric sterility before inoculation is shown in Figure 2 for C. difficile and in Figure 3 for S. aureus. Results suggest that the autoclaving method only seems effective when checking the presence of microorganisms with the viable plate counting method. However, when qPCR is used for the presence of microorganisms, there are positive results for both tested microorganisms. Although autoclaving eliminates all viable microorganisms, the DNA is not removed from the textile fabric. This in turn leads to some potentially important issues. Firstly, is the use of qPCR or any other method relying on DNA presence detection appropriate for assessment of specific microbial presence or count? Our results clearly show that the positive result for presence of DNA does not necessarily mean viable bacteria. This is especially important when considering hospital textiles for multiple uses. The second important issue is the presence of leftover DNA after the cleaning process. On the other hand, our results have clearly shown that the method of soaking of inoculated textiles in sodium hypochlorite solution leaves no viable microorganisms or their DNA. These results strongly support the use of disinfectants, which are, for the removal of microorganisms, more efficient than heat alone.

Figure 2.

Efficiency of autoclaving textiles swatches inoculated with C. difficile (at); soaking textiles swatches inoculated with C. difficile in sodium hypochlorite solution (sh); and confirmation of fabric sterility before inoculation (s). qPCR: real time polymerase chain reaction.

Figure 3.

Efficiency of autoclaving textile swatches inoculated with S. aureus (at); soaking textile swatches inoculated with S. aureus in sodium hypochlorite solution (sh); and confirmation of fabric sterility before inoculation (s). qPCR: real time polymerase chain reaction.

The efficiency of sterilization of fabric before inoculation is confirmed by the results in Figures 2 and 3, which show no presence of microorganisms detected with either conventional or molecular methods.

Comparison of the standard versus molecular methods detection efficiency for textile sampling

A comparison of the detection of the four challenge pathogens on textiles by conventional and molecular techniques is shown in Figures 4 –7 and the exact cfu or cevs of detected microorganisms/spores are shown in Table 3. Values of retrieved microorganisms from inoculated textiles were highest when microorganisms were detected with qPCR; the second most efficient method was viable plate counting after the elution method and the less efficient method was sampling with RODAC agar plates. Results show that the elution method is much more effective in comparison with RODAC agar plates, which also 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.¹⁸

Figure 4.

Efficiency of conventional and molecular methods for detecting C. difficile spores on textiles. Initial spore concentration on textile pieces before overnight drying: (a) 2.8 × 10⁷cfu/ml; (b) 1.0 × 10⁵cfu/ml; (c) 5.2 × 10⁴cfu/ml. qPCR: real time polymerase chain reaction.

Figure 5.

Efficiency of conventional and molecular methods for detecting S. aureus on textiles. Initial bacterial concentration on textile pieces before overnight drying: (a) 6.2 × 10⁷cfu/ml; (b) 5.5 × 10⁴cfu/ml; (c) 3.0 × 10³cfu/ml. qPCR: real time polymerase chain reaction.

Figure 6.

Efficiency of conventional and molecular methods for detecting K. pneumoniae on textiles. Initial bacterial concentration on textile pieces before overnight drying: (a) 5.0 × 10¹⁰cfu/ml; (b) 5.0 × 10⁸cfu/ml; (c) 1.7 × 10⁷cfu/ml; (d) 5.6 × 10⁴cfu/ml. qPCR: real time polymerase chain reaction.

Figure 7.

Efficiency of conventional and molecular methods for detecting P. aeruginosa on textiles. Initial bacterial concentration on textile pieces before overnight drying: (a) 1.6 × 10⁹cfu/ml; (b) 9.0 × 10⁸cfu/ml; (c) 3.4 × 10⁴cfu/ml. qPCR: real time polymerase chain reaction.

Table 3.

Mean cell equivalent values detected with real time polymerase chain reaction (qPCR) and Ct values, mean colony forming unit (cfu) values of detected microorganisms/spores with elution method and RODAC agar plates

qPCR [cev/ml] / Ct	Elution [cfu/ml]	RODAC [cfu]
Clostridium difficile
a	5.29E + 06 / 23.34	7.00E + 04	9.80E + 01
b	47.43E + 02 / 28.52	2.47E + 02	3.83E + 01
c	9.50E + 00 / 34.26	0.00E + 00	5.00E + 00^a
Staphylococcus aureus
a	5.52E + 04 / 27.88	1.55E + 04	>3.00E + 02
b	8.69E + 02 / 34.04	3.33E + 00	8.47E + 01
c	1.20E + 02 / 34.39	0.00E + 00	1.33E + 00^a
Klebsiella pneumoniae
a	5.91E + 07 / 22.26	3.60E + 08	>3.00E + 02
b	1.49E + 06 / 29.09	2.40E + 07	1.00E + 00^a
c	6.92E + 03 / 36.55	0.00E + 00	0.00E + 00
d	2.35E + 04 / 35.46	0.00E + 00	0.00E + 00
Pseudomonas aeruginosa
a	5.92E + 06 / 27.99	1.00E + 04	>3.00E + 02
b	5.79E + 05 / 31.51	0.00E + 00	0.00E + 00
c	4.12E + 04 / 31.96	0.00E + 00	0.00E + 00

Not in statistically accurate plate count range.

The cfu captured from the inoculated textiles were lower than the initial applied concentration of microorganisms, which was the case for all four sampled microorganisms. We assume that with the elution method, all live and uninjured microorganisms were detected and that a certain percentage of microorganisms did not survive the procedure of overnight drying. This explains that the cfu captured from the inoculated textiles with elution were lower than the initial applied concentration of microorganisms for all four sampled microorganisms. This is also consistent with the findings of Cody et al.¹⁸ that 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. The downside of this method is that it belongs to a group of destructive methods, where a piece of textile 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. Therefore, this method can be particularly useful under laboratory criteria; however, its use in a real environment is limited.

Sampling textiles with RODAC agar plates provides much less effective and also less reliable results. This was particularly the case when textiles inoculated with higher concentrations of microorganisms were sampled, which is shown in Figure 5: sample a; Figure 6: sample a; and Figure 7: sample a, where the result for cfus captured from inoculated textiles is over 300, which leads to inability to count on the RODAC agar plate. 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.¹⁹ If surfaces are rough or wet, then the sampling is inaccurate or the resulted growth on the agar may be confluent.¹⁹ Another problem when using RODAC agar plates for sampling is that the efficiency of this method depends on the evenness of the surface tested.²⁰ Because the structure of a textile is a mechanical combination of warp and weft, only a small contact area is available, which leads to low cfu counts. This is clearly seen in Table 3 at sample c for C. difficile, where only five cfus were obtained on the RODAC agar plate, at sample c for S. aureus, where only one point three cfus were obtained, and at sample b for K. pneumoniae, where only one cfu was obtained. The general ranges in common acceptance for countable numbers of colonies on a plate are between 30 and 300. The origin of those ranges is worth examination.²¹ The low counts of microorganisms covered by the RODAC plate technique appear in several studies.²² Eriksson et al.²³ also concluded in their research that the contact plate technique is inappropriate for determination of bioburden on textiles.

Considering the common acceptance for countable numbers of colonies on a plate, sample c in Figure 4, sample c in Figure 5, samples c and d in Figure 6 and samples b and c in Figure 7 do not give any results with both conventional techniques, but do clearly give the results with molecular techniques (qPCR). This leads to the conclusion that there were no viable microorganisms after the overnight drying process, since viability in bacteria is synonymous with the ability to form colonies on a solid growth medium and to proliferate in liquid nutrient broths.²⁴ Accurate determination of live, dead and total bacteria is important in many microbiological applications, but cannot be obtained with traditional, culture-based tests, which are in addition also time-consuming and can work poorly with slow-growing or viable but non-culturable (VBNC) organisms.²⁴ The solution is clearly shown in our results, where qPCR gives positive results even where conventional techniques do not give any results. This can indicate the presence of VBNC cells or injured bacteria, which cannot be cultured on standard bacteriological media,²⁵ but which remain morphologically intact.^26–30 They can still be active cells, and several authors^27,28,30,31 have shown that non-culturable bacteria can be metabolically active and that non-culturable, pathogenic bacteria can maintain their infectivity.^28,32 Investigated microorganisms can form VBNC cells.³³ It has also been demonstrated that genes encoding virulence determinants are retained for long periods in cells that have lost culturability.³⁴ Although the ability of VBNC forms to cause disease has not been absolutely demonstrated, non-culturable pathogens may still pose a hazard to public health.³⁵ Non-culturable cells of pathogens are capable of expressing virulence factors, such as toxins, in response to exogenous stimuli.³⁶

The positive results with qPCR even where conventional techniques do not give any results can also indicate the presence of live microorganisms that were not detected with conventional methods or dead microorganisms, since DNA can persist in a PCR-detectable form in actively killed cells for significant periods of time.³⁷ Pote et al.³⁸ have shown that leftover of free DNA has the potential for biologically active DNA to be transported over considerable distances in water-saturated soil and ground water in continuous-flow and transform bacteria to confer antibiotic resistance by natural mixing of DNA in the subsurface environment . Another study³⁹ also reports the existing link between antibiotic use and the significant increase in drug-resistant human bacterial pathogens where hot spots for gene transfer in the soil/plant environment, for example manured soil, have been identified as reservoirs of resistance genes. One of the most important of all is the use of antibiotics in agriculture, where resistance genes occurring in gut commensal and pathogenic bacteria of intensively reared farm animals can spread to the soil bacterial community.³⁹ The question is if the transfer of resistant genes as leftovers of free DNA after the textile cleaning process can be transferred to the non-resistant bacteria of a patient in the case of multiple-use of hospital textiles by similar transport as occurs in soil or ground water.

Although molecular methods, such as PCR, are widely used especially in clinical microbiology, the methods have not yet been transferred for verification of textile hygiene. Our study demonstrated that molecular methods can be very useful for detecting nosocomial pathogens on textiles and also offers a possibility to confirm the presence of microorganisms in states that cannot be detected by conventional sampling techniques. Further work will be focused on testing the method on samples from the hospital environment, such as bed clothes and pajamas.

Footnotes

Funding

This work was supported by the Slovenian Research Agency ARRS (project No.1000-10-310152).

Acknowledgments

We thank the company Omega Ltd. for kindly lending us a demo unit of StepOne Real Time PCR System (Applied Biosystems). We also thank the Institute of Public Health Maribor for supplying the bacterial strains.

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