Assessment of sample handling practices on microbial activity in sputum samples from patients with...

ORIGINAL ARTICLE

Assessment of sample handling practices on microbialactivity in sputum samples from patients with cysticfibrosisA. Nelson1, A. De Soyza2, S.J. Bourke3, J.D. Perry4 and S.P. Cummings1

1 School of Applied Sciences, Ellison Building, University of Northumbria, Newcastle upon Tyne, UK

2 Lung Transplantation and Immunobiology Group, Newcastle University and the Freeman Hospital Newcastle upon Tyne, UK

3 Adult Cystic Fibrosis Unit, Department of Respiratory Medicine, Royal Victoria Hospital, Newcastle upon Tyne, UK

4 Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, UK

Introduction

The airways of patients with cystic fibrosis (CF) have

been shown by both culture-dependent (Baltimore et al.

1982; Tunney et al. 2008) and independent methods,

which do not require prior cultivation of micro-organ-

isms, (van Belkum et al. 2000; Rogers et al. 2003) to be a

complex microbial environment containing many differ-

ent taxa. Analysis of these microbial communities by

culture-dependent methods shows that patients with CF

are most likely to suffer from infections caused by Pseudo-

monas aeruginosa, Staphylococcus aureus and Haemophilus

influenzae, respectively (Cystic fibrosis foundation, patient

registry 2007). Culture-independent studies are largely in

agreement with culture; however, molecular studies also

show the increased prevalence of Prevotella spp., Neisseria

spp. and oral streptococci such as the Streptococcus milleri

group (Harris et al. 2007; Sibley et al. 2008). Comparisons

of culture-dependent and independent techniques have

highlighted the limitations of routine microbial culture to

unearth the true diversity of this environment. This may be

attributed to the fastidious nature of many anaerobic bac-

teria (Bittar et al. 2008) or attributed to heavy growth of

Ps. aeruginosa during aerobic culture making isolation of

clinically significant organisms difficult. Whilst validation

and the improving selectivity of routine culture still make

this the ‘gold standard’ for identification of many CF

isolates, the use of culture-independent techniques is

Keywords

DGGE (denaturing gradient gel

electrophoresis), fungi, Pseudomonads.

Correspondence

Stephen P. Cummings, School of Applied

Sciences, Ellison Building, University of

Northumbria, Newcastle upon Tyne NE1 8ST,

UK. E-mail:

[email protected]

2010 ⁄ 0670: received 22 April 2010, revised

11 June 2010 and accepted 14 June 2010

doi:10.1111/j.1472-765X.2010.02891.x

Abstract

Aim: The aim of this study was to quantitatively and qualitatively assess the

effect of sample storage on the metabolically active microbial community found

in sputum samples from patients with cystic fibrosis (CF).

Methods: Sputum samples were collected and split in two equal aliquots one of

which was immersed in RNAlater and refrigerated immediately, the second

stored at room temperature for 24 h and RNAlater was subsequently added.

mRNA was extracted, and RT-PCR-DGGE and qPCR analysis of the bacterial

and fungal communities was carried out.

Results: Significant differences in the bacterial communities between the two

protocols were observed but there were no significant difference seen in the fun-

gal community analyses. Analysis by qPCR demonstrated that room temperature

storage gave statistically significant increases in eubacteria and Pseudomonas spp.

and a statistically significant decrease in those of Haemophilus influenzae.

Conclusions: The analysis of metabolically active microbial communities from

CF sputum using molecular techniques indicated that samples should be stored

at 4�C upon addition of RNAlater to obtain an accurate depiction of the CF

lung microbiota. Also, storing respiratory samples at room temperature may

cause an over representation of Pseudomonas aeruginosa and mask the presence

of other clinically significant organisms.

Letters in Applied Microbiology ISSN 0266-8254

272 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 272–277

ª 2010 The Authors

becoming increasingly important as a clinical tool. There-

fore, it is important to assess the effects of sample storage

on microbial nucleic acids because many studies are multi-

centre and require shipping of samples from the ward to

the laboratory which may be on different sites.

The effect of sample handling and storage on the

results of routine microbial culture has previously been

assessed. It has been demonstrated that storage of samples

at 4�C for 48 h produces reproducible results by culture

in up to 75% of samples (Gould et al. 1996). A recent

study showed that these results held true for the predomi-

nant organism in samples when cultured immediately vs

samples stored at 4 and at 20�C (room temperature) (Pye

et al. 2008). However, quantitative culture of the samples

demonstrated that in 24% of samples stored at 4�C there

were at least 10-fold fewer viable organisms compared to

8% in the samples stored at 20�C (Pye et al. 2008). The

findings of Pye et al. (2008) recommends storage of

samples at room temperature rather than 4�C, which is

now becoming common practice in many studies using

both culture-dependent and independent methods. The

effect of sample storage on the results of molecular stud-

ies is yet to be defined which may have a significant effect

on the results of such studies, especially when looking at

metabolically active members by way of mRNA because

of the short half-life of this molecule.

In this study, we hypothesized that storing samples at

room temperature skews the data set from what would

truly be seen in the CF lung. We propose a simple

method for processing sputum samples to give a more

accurate depiction of how the microbial community is

composed in-vivo.

Methods

Collection of sputum samples

Five spontaneously expectorated sputum samples were

collected from adult patients attending a CF clinic and

were immediately divided into two equal aliquots. One set

of aliquots were immediately treated with RNAlater�

(Ambion, Austin, TX), which is bacteriostatic and stored

at 4�C. The second set of aliquots were initially incubated

at room temperature (20�C) for 24 h. After 24 h, these

aliquots were treated with RNAlater� (Ambion), and RNA

extraction was then performed on all aliquot samples.

Ethical approval was obtained from County Durham

and Tees Valley research ethics committee REC (ref

07 ⁄ H0908 ⁄ 68).

Extraction of mRNA and PCR amplification

The mRNA was extracted from sputum and seven refer-

ence organisms (Fig. 1a). The samples were washed with

phosphate-buffered saline to remove any contamination

from oral flora (Rogers et al. 2006). Sputasol (Oxoid) was

added and mRNA was extracted (MoBio Ultraclean

Microbial RNA; MoBio Laboratories, Solana Beach, CA)

from a 1Æ8 ml aliquot according to the manufacturer’s

instruction. Reverse transcription was performed with

–0·8 1–0·6 –0·4 –0·2 0 0·80·60·40·2

–0·5

–1

–1·5

0·5

1

1·5

Patient 1-fridge

Patient 2-fridgePatient 3-fridge

Patient 4-fridge

34% Inertia

21% Inertia

PC 2

PC 1

Patient 1-RTPatient 2-RT

Patient 3-RT

Patient 4-RT

Patient 5-RT

Patient 5-fridge

0

(b)(a)H. influenzae

S. aureus

Pseudomonasaeruginosa

Burkolderia spp./Steno. maltophilia

R. pickettii

Achromobacterxylosoxidans

Patient number; 1 2 3 4

Fridge Room temperature5 1 2 3 4 5

Figure 1 (a) Denaturing gradient gel electrophoresis (DGGE) analysis showing the effect of storage on the bacterial communities of the five

patients with cystic fibrosis (CF). The bands indicated in the far left lane are derived from reference strains and were used to give a putative identi-

fication to bands in the samples that may indicate common CF isolates (b) Principle component analysis of the bacterial DGGE profiles generated

in Fig. 1a.

A. Nelson et al. Sample handling of CF sputum

ª 2010 The Authors

Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 272–277 273

Superscript II reverse transcriptase (Invitrogen) using

random hexamers (Qiagen, Hilden, Germany) and 40 U

RNaseOUT� (Invitrogen, Carlsbad, CA) according to the

manufacturer’s instructions.

PCR amplification of the V3 region of the bacterial 16S

rRNA gene was performed with primers V3fc and V3r as

described by Muyzer et al. (1993) according to the meth-

ods of Baxter and Cummings (2006). The fungal commu-

nity of sputum was amplified using the PCR primers U1

and U2-GC (Sandhu et al. 1995) which are specific for

the fungal 28S rRNA gene. The reaction was performed

with 0Æ5 lmol l)1 each primer, 2X amplification buffer,

1X PCR enhancement solution, 0Æ3 mmol l)1 each dNTP,

1 mmol l)1 MgSO4, 500 mg BSA, 1Æ25 U Pfx Platinum

polymerase (Invitrogen) and 1 ll cDNA template made

up to 50 ll with sterile 18Æ2 X H2O. The cycling condi-

tions used were an initial denaturation for 5 min then 10

cycles of 94�C for 1 min, 60�C ()1�C per cycle) for

1 min and 68�C for 30 s followed by 25 cycles of 94�C

for 1 min, 50�C for 1 min and 68�C for 30 s with a final

extension at 68�C for 10 min.

Denaturing gradient gel electrophoresis (DGGE)

DGGE analysis was performed using the D-Code DGGE

system (Bio-Rad, Hercules, CA). For analysis of bacterial

populations, PCR products were loaded on to polyacryl-

amide gels (12%) with a denaturant gradient of 35–65%

denaturant (with 100% denaturant corresponding to

7 mol l)1 urea plus 40% v ⁄ v formamide). Gels were run at

60�C for 4Æ5 h at 200 V, analysis of the fungal community

was performed with a denaturing gradient of 25–55% at

70 V for 17 h. Gels were stained with SYBR Green I (Invi-

trogen) and viewed with UV transillumination using the

Gel Doc 2000 gel documentation system (Bio-Rad).

Specific DGGE bands were excised and eluted in 10 ll

of molecular biology grade water overnight. The eluted

DNA was amplified, and the products were sequenced

using BigDye� Terminator cycle sequencing kit (Applied

Biosystems, Foster City, CA) and sequenced using ABI

Prism� 3130 Genetic Analyzer (Applied Biosystems, Foster

City, CA).

Real-time PCR (qPCR)

The qPCR method used herein was as described (Baxter

and Cummings 2008). Briefly, a plasmid standard was

constructed containing the target region for each primer

set using DNA extracted from the appropriate control

strain. Standard curves were prepared for each primer set

using triplicate 10-fold dilutions of the plasmid standard

to contain the target sequence at 300 000–30 copies ml)1.

The RNA from the samples was reverse transcribed to

cDNA as described previously, and the cDNA was used as

template in the qPCRs. The cycling conditions used were

an initial enzyme activation step at 95�C for 15 min, then

50 cycles of 95�C for 10 s, annealing temperature

(Table 1) for 15 s and extension at 72�C for 20 s on

RotorGene 3000 instrumentation (Corbett Life Sciences,

Sydney, Australia). Target copy numbers of the gene of

interest (Table 1) for each reaction, performed in tripli-

cate, were calculated from the standard curve and were

used to ascertain the number of copies of the target gene

(Table 1) per ml of sputum.

Statistical analyses

Analysis of the DGGE banding patterns was performed

using Quantity One� software (v4.1.1.; Bio-Rad). Prin-

ciple component analysis was performed to assess the var-

iance in the dataset using PAST (Hammer et al. 2001),

and the significance (P < 0Æ05) of the first principle com-

ponent was determined by general linear model anova

using Minitab� 15. For real-time PCR, all data were nor-

malized, and a paired Student’s t-test of the cohorts from

4�C and room temperature was performed using Mini-

tab� 15 (v1.30.0.) for each of the taxa.

Results

DGGE analysis of microbial communities

The DGGE analyses of the bacterial community produced

32 distinct band positions (12–22 bands per lane).

Qualitative observation showed that refrigerated samples

Table 1 Primers used for qPCR assay

Primer Sequence Target gene

Annealing

temperature (ºC) Reference

Eub 338 ACT CCT ACG GGA GGC AGC AG 16S rRNA 65 Lane 1995

Eub 518 ATT ACC GCG GCT GCT GG Muyzer et al. 1993

Ps-f GRM CGC TAA TAC CGC NTA CGT 16S rRNA 50 Baxter and Cummings 2008

Ps-r TCC TCT CAG ACC AGT TAM GGA

HI-IV ACT TTT GGC GGT TAC TCT GT Outer membrane

protein P-6

55 van Ketel et al. 1990

HI-V TGT GCC TAA TTT ACC AGC AT

Sample handling of CF sputum A. Nelson et al.


ª 2010 The Authors

had more bands in the top third of the gel (lower GC

content) compared to those stored at room temperature

(Fig. 1a). Statistical analysis of the band profiles demon-

strated that samples were significantly grouped

(P < 0Æ001) according to the collection protocol along the

first principle component, explaining 34% of the variance

(Fig. 1b). The refrigerated sputum samples show a greater

scatter by the second principle component, explaining

21% of the variance, than the samples stored at room

temperature.

DGGE analysis of the fungal community gave a total of

17 distinct bands (4–10 per lane). There was no signifi-

cant difference between sputum collection protocols but

the band intensities were less in the samples that were

stored at room temperature when compared to those that

were refrigerated (Fig. 2). Four bands (F1–F4) were

excised, sequenced and deposited in GenBank (accession

numbers GU001640, GU065334–36, respectively). The

NCBI BlastN tool was used to search for closest depos-

ited sequence match. The closest related sequences were

Candida dubliniensis (FM992695Æ1) 99%, Candida albicans

(GQ495089Æ1) 100%, Candida parapsilosis (AY497686Æ1)

99% and Aspergillus fumigatus (FM197606Æ1) 99%, respec-

tively.Real-time PCR results

Quantification of metabolically active total bacteria and

Pseudomonas spp. using the mean copy number of the

16S rRNA gene indicated a greater than twofold increase

in Pseudomonas spp. 16S rRNA gene in the samples

stored at room temperature compared to the refrigerated

samples (Fig. 3). This finding was statistically significant

for both taxa (P < 0Æ001). In contrast, quantification of

the mean H. influenzae P6 outer membrane protein copy

number revealed a >50% decrease in P6 gene copy num-

bers in the samples stored at room temperature compared

to the sample stored at 4�C that was statistically signifi-

cant (P < 0Æ001).

The log of the mean eubacterial copy number per ml

for the samples stored at 4�C was 9 (range 7Æ41–9Æ79), for

Pseudomonas spp. the mean copy number was 7Æ10 (range

6Æ53–8Æ79) and for H. influenzae the mean copy number

was 3Æ87 (range 3Æ61–4Æ28).

Discussion

Many studies on CF therapies are multicentre and

involve both qualitative and quantitative bacterial analy-

sis. This usually entails shipping of samples from study

sites to a central processing laboratory. Here, we show

that the storage of sputum samples can have a powerful

effect on the results of culture-independent techniques

for detection of bacteria. In most situations, it is not

feasible or in some cases possible to process sputum sam-

ples immediately especially if they need to be transported

F2F1

F3

F4

Patient number; 1 2 3 4 5 1 2 3 4 5

Fridge Room temperature

Figure 2 Denaturing gradient gel electrophoresis analysis showing

the effect of sample storage on the fungal communities present in

the sputum samples from the five patients with cystic fibrosis. F1–F4

are bands that were subsequently excised and sequenced.

*

0·8

0·7

0·6

* *

0·5

0·4

0·3

0·2

Nor

mal

ised

dat

a

Eubacteria Pseudomonas spp.

Haemophilus influenzae

0·1

0

Figure 3 Normalized qPCR data to demonstrate the mean effect of

sample storage on bacterial metabolic activity. Grey bars indicate

samples stored at 4�C; White bars indicate samples stored at room

temperature. *indicates the difference between sample storage is

statistically significant (P < 0Æ05).


ª 2010 The Authors


from the ward to the laboratory or from one site to

another. In this study, we take samples that have been

immersed in RNAlater and stored at 4�C to be an accu-

rate representation of the microbial community as it

would be observed in the lung. We have based this

assumption on the preservative property of RNAlater

preventing RNA degradation as well as the bacteriostatic

properties it possesses preventing any change in the RNA

profile of the sample. DGGE analysis of the bacterial

community indicated that storage of samples significantly

affected the bacterial community in the sputum

(P < 0Æ001). It appears from the change in the DGGE

profiles that storage at 4�C is more favourable for visual-

izing organisms with a lower 16S rRNA GC content

(Fig. 1a). Statistical analysis of the profiles showed that

the samples stored at room temperature clustered

together more along the second principle component

than the samples stored at 4�C, suggesting that storage at

room temperature masks intersample variability (Fig. 1b).

Culture-independent techniques have previously been

shown to identify many more taxa in CF sputum samples

than the corresponding sample analysed using culture-

dependent techniques our data supports those of

previously published work (van Belkum et al. 2000).

Moreover, quantification using qPCR data showed a

significant increase (P < 0Æ001) in the number of total

bacteria and Pseudomonas spp. in the sputum samples

stored at room temperature (Fig. 3.). These observations

may indicate that growth of some microbial taxa, par-

ticularly Ps. aeruginosa, is favoured at room temperature

in comparison with 4�C. The increase in numbers of

Pseudomonas spp. after storage at room temperature may

explain the overgrowth of Ps. aeruginosa, commonly seen

in routine microbiology. This might result in the clinical

significance of Ps. aeruginosa being overestimated in such

samples. It was also shown that H. influenzae numbers

are significantly decreased after storage at room tempera-

ture when compared to storage at 4�C (P < 0Æ001). It has

been shown previously that H. influenzae has been difficult

to recover from transport swabs because of its fastidious

nature (Rishmawi et al. 2007). It has also been observed

that H. influenzae is more difficult to recover from

sputum samples after postage than from fresh samples

processed immediately (May and Delves 1964). However,

our data contradict those of Pye et al. (2008) who suggest

that storage at room temperature produces more favour-

able conditions for the recovery of H. influenzae.

The numbers of active bacteria present in CF sputum

have not previously been assessed using culture-indepen-

dent methods. Using culture-dependent techniques,

between 108 and 109 CFU ml)1 were observed for the

predominant organism in the sample (Pye et al. 2008).

In contrast, the total bacterial 16S rRNA copy number

per ml in our sample set is between 107 and 109 with a

mean of 1Æ00 · 109 and between 106 and 107 copies per

ml of this relates to Pseudomonas spp. With most

organisms having more than one copy of this gene, our

account of the active bacteria in CF sputum is signifi-

cantly lower than the number of viable cells as deter-

mined by culture when using this gene as a target. Pye

et al. (2008) also showed that H. influenzae was present

at 109 CFU ml)1 when it is the predominant organism,

whereas H. influenzae was not isolated by culture (data

not shown) from any of our patient cohort but was

identified using molecular techniques. This highlights

the increased sensitivity of molecular techniques

compared to culture which supports the findings from

previous studies (van Belkum et al. 2000; Dalwai et al.

2007).

There has been no previous work carried out by

culture-dependent or independent methods showing the

effect of storage on fungi from CF sputum. The results of

this study show that, although there was a slight decrease

in band intensity for fungi when stored at room tempera-

ture, there was no significant difference between the

sample collection procedures. Because of there being a

slightly lower band intensity for the samples stored at

room temperature, our data suggest that samples required

for fungal analysis should be stored at 4�C to preserve the

nucleic acids in the sample. Sequence analysis of DGGE

bands allowed the identification of A. fumigatus, a fungal

pathogen that is known to adversely effect lung function

(Amin et al. 2010). There is currently very little informa-

tion pertaining to the fungal members of the CF lung

microbiota and how they persist. Further work is required

to fully analyse this community.

In conclusion, this study supports handling protocols

where respiratory samples being used in molecular studies

should have RNAlater added immediately and then to be

subsequently stored at 4�C until required for processing if

an accurate depiction of the community is to be observed

and accurate quantification of bacterial numbers is to be

achieved. However, for fungi, sample handling procedures

are less crucial for quantification.

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