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Previous Article | Next Article 
Journal of Clinical Microbiology, January 2000, p. 351-353, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simplified Protocol for Pulsed-Field Gel
Electrophoresis Analysis of Streptococcus
pneumoniae
M. Catherine
McEllistrem,1,2,*
Janet E.
Stout,3 and
Lee H.
Harrison1,2
Public Health Infectious Diseases Laboratory,
University of Pittsburgh Graduate School of Public
Health,1 Department of Medicine,
University of Pittsburgh School of Medicine,2
and Veterans Affairs Medical
Center,3 Pittsburgh, Pennsylvania
Received 23 June 1999/Returned for modification 19 August
1999/Accepted 8 October 1999
 |
ABSTRACT |
A variety of pulsed-field gel electrophoresis (PFGE) protocols for
the molecular subtyping of Streptococcus pneumoniae have been reported; most are time-consuming and complex. We sought to modify
reference PFGE protocols to reduce the time required while creating
high-quality gels. Only protocol modifications that resulted in
high-quality banding patterns were considered. The following protocol
components were modified. Lysis enzymes (lysozyme, mutanolysin, and
RNase A) were deleted in a stepwise fashion, and then the lysis buffer
was deleted. Lysis and digestion were accomplished in a single step
with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3)
incubation at 50°C in the absence of proteinase K. All enzymes except
the restriction enzyme were omitted. A minimum incubation time of
6 h was required to achieve high-quality gels. All of the
reactions were performed within 9 h, and the total protocol time
from lysis to gel completion was reduced from 3 days to only 36 h.
Combining lysis and digestion into a single step resulted in a
substantial reduction in the time required to perform PFGE for S. pneumoniae. The ES solution may have caused cell lysis by
activating N-acetylmuramyl-L-alanine amidase,
the pneumococcal autolysin.
| |
INTRODUCTION |
Molecular subtyping has
revolutionized the field of infectious disease epidemiology. Molecular
epidemiology allows the differentiation of isolates that appear
identical by conventional methods, such as antimicrobial susceptibility
testing or serotyping. While a variety of subtyping techniques have
been developed, an ideal method would be simple to perform, be
reproducible, and result in high-resolution banding patterns.
Streptococcus pneumoniae is a significant public health
concern, especially with the emergence of drug-resistant strains. Molecular epidemiology can be used to study the spread of
drug-resistant S. pneumoniae in populations, making it an
important adjunct for surveillance of this important pathogen.
Pulsed-field gel electrophoresis (PFGE), created by Schwartz, Cantor,
and colleagues in 1982 (22), allows large DNA fragments to
be separated on an agarose gel by virtue of their molecular weights.
The electric field is sequentially changed at variable time intervals,
or pulse times. The larger fragments take longer to realign in each
field and thus move a shorter distance down the gel compared to the
lower-molecular-weight fragments. Since the inception of PFGE,
modifications have allowed the development of both field
inversion gel electrophoresis (FIGE) and contour-clamped homogenous
field electrophoresis (CHEF). CHEF has hexagonally arranged
electrodes which cause movement of DNA fragments down a gel by
alternating pulsed currents (21).
Commonly used methods for the molecular subtyping of S. pneumoniae include PFGE, BOX fingerprinting, restriction fragment end labeling, ribotyping, and PCR with primer enterobacterial repetitive intergenic consensus sequence (ERIC2) (11).
While the first three provide the most discrimination between strains, it has been suggested that BOX fingerprinting and restriction fragment
end labeling are the best methods because of the quick turnaround time
and ease of computer analysis. Restriction fragment end labeling can be
completed in 48 h, while BOX fingerprinting requires 72 h to
perform. Recently, amplified-fragment length polymorphism analysis
(AFLP) has been compared to PFGE in terms of time to completion and
dendrogram analysis. While variation occurred in the dendrogram
clusters, both protocols required approximately 2.5 days to complete,
in addition to the 20 h required to perform PFGE (25).
PFGE has been criticized for being time-consuming and labor-intensive.
We sought to simplify existing S. pneumoniae PFGE protocols
so as to create reproducible, high-quality gels with minimal time and effort.
| |
MATERIALS AND METHODS |
A Medline search was performed to identify S. pneumoniae PFGE protocols published from 1985 to 1998 (2, 3,
5, 6, 9, 13-17, 19, 23, 25, 26). Most protocols contained the
following steps: bacterial cell suspension and agarose suspension, lysis, digestion, Tris-HCl-EDTA (TE) washes, enzyme restriction, and
electrophoresis. We serially modified aspects of several published protocols to create a simplified method. Only changes which resulted in
reproducible high-quality banding patterns were adopted. The standard
protocol we used was as follows.
S. pneumoniae cultures were grown overnight on Trypticase
soy agar with 5% sheep blood (BBL) and suspended in 2 ml of cell suspension buffer (1 M NaCl-10 mM Tris-HCl [pH 7.6]) to an optical density of 1.3 to 1.5 at 450 nm. The bacterial suspension was mixed
with an equal amount of 2% low-melting-point agarose (Sea Plaque; FMC
Bioproducts, Rockland, Maine) and pipetted into 100-µl plug molds.
After being solidified on ice for 10 min, the plugs were lysed by
incubation with 2 ml of lysis buffer (1 M NaCl, 100 mM EDTA, 6 mM
Tris-HCl, 0.5% Brij 58, 0.5% deoxycholate, 0.5% N-lauroyl
sarcosine [pH 7.6]) supplemented with 1 mg of lysozyme/ml-50 µg of
RNase A/ml for 3 h at 37°C. Next, each plug was incubated with 2 ml of ES buffer (0.5 M EDTA-1% N-lauroyl sarcosine [pH 8.0 to 9.3]) and 100 µg of proteinase K/ml overnight at 50°C. The
plugs were then washed three times with 10 ml of TE buffer containing
10 mM Tris-HCl and 1 mM EDTA (pH 7.6) at 37°C for 30 min each time.
After preincubation of a 2- by 5-mm section of a plug in NE 4 buffer
(New England Biolabs, Inc., Beverly, Mass.) for 20 min, the DNA was
digested with NE 4 buffer mixed with 30 U of SmaI and 200 µg of bovine serum albumin/ml at room temperature overnight. For ease
of comparison, the run parameters from the modified protocol were used
(5, 6, 13, 14, 16, 17, 22, 25, 26). The total time from
lysis to gel completion was 3 days.
We performed PFGE on invasive penicillin-nonsusceptible S. pneumoniae (PNSP) strains from the Baltimore metropolitan area, collected as part of the Maryland Bacterial Invasive Disease
Surveillance project (BIDS) (10). BIDS is a component of the
multistate Emerging Infections Program that is coordinated by the
Centers for Disease Control and Prevention (CDC). Serotypes were
determined by the quellung reaction with type-specific antiserum
prepared at the CDC (4). Strains of serotypes 6A, 9V, 14, 19A, 19F, and 23F were chosen to provide a representative sample of
PNSP isolates (12). Penicillin-susceptible isolates were
also chosen from serotypes 9V, 23F, and 33F. A serotype 23F isolate,
the multiresistant S. pneumoniae Spanish clone (Cleveland
strain), was donated by A. Tomasz of Rockefeller University, New York,
N.Y. (20).
| |
RESULTS |
Serial deletion of the lysis enzymes, including lysozyme,
mutanolysin, and RNase A, caused no qualitative or quantitative changes
in the banding pattern. The lysis step was deleted, and the digestion
step was successfully performed in the absence of proteinase K. Lysis
and digestion were completed in a single step with ES buffer (pH 8.0 to
9.3) at 50°C for 6 h. After the plug was washed three times in
TE buffer, the DNA was digested by a 2-h incubation with 250 U of
SmaI/ml mixed in NE 4 restriction buffer supplemented with
200 µg of bovine serum albumin/ml. The total protocol time from lysis
to the end of electrophoresis was reduced to 36 h. While melting
the agarose containing the DNA fragments at 62°C for 10 to 15 min is
technically easier (13, 25), this resulted in sporadic loss
of high-molecular-weight bands, increased lane background, and
decreased overall intensity of the remaining bands.
Incubation with ES buffer with or without proteinase K
(1 mg/ml) for less than 6 h did result in an identical
banding pattern compared to that with the standard protocol. However,
the bands were faint due to increased lane background, making
interpretation more difficult. The addition of a second block of time
to the gel run parameters allowed the improved resolution of bands
between 50 and 100 kb and facilitated analysis of the gel. We did note that a small percentage of the organisms did not lyse during the first
6-h incubation; however, when these isolates were incubated a second
time in the ES buffer overnight, adequate lysis did occur. The
likelihood of complete lysis increased if we incubated only one plug in
each tube of ES buffer.
We were able to reproduce this simplified protocol in two laboratories.
The plugs containing pneumococcal DNA still create reproducible banding
patterns after 6 months of storage in TE buffer at 4°C. The final
simplified protocol is as follows. S. pneumoniae cultures
were grown overnight on Trypticase soy agar with 5% sheep blood (BBL)
and suspended in 2 ml of cell suspension buffer (1 M NaCl-10 mM
Tris-HCl [pH 7.6]). The cell suspension was adjusted to an optical
density of 1.3 to 1.5 at 450 nm. Equal amounts of bacterial suspension
and 2% low-melting-point agarose (Sea Plaque) were mixed and pipetted
into 100-µl plug molds. After the plugs were solidified on ice for 10 min, they were lysed and digested in one step. Each plug was incubated
in 2 ml of ES buffer (pH 8 to 9.3) for 6 h at 50°C. The plugs
were then washed three times with 10 ml of TE buffer containing 10 mM
Tris-HCl and 1 mM EDTA (pH 7.6) at 37°C for 15 min each time. After
preincubation of a 2- by 5-mm section of a plug in NE 4 restriction
buffer for 15 min, the DNA was digested with NE 4 buffer mixed with 250 U of SmaI/ml and 200 µg of bovine serum albumin/ml at room
temperature for 2 h. Each 1- by 5-mm section of plug was loaded
into a 1% agarose gel, and PFGE was performed with CHEF DRIII at the
following parameters: pulse times, 1 to 30 s for 19 h and 5 to 9 s for 8 h; 198 V; 1 liter/min; 14°C. The gel was
stained with ethidium bromide for 20 min and captured on the Bio-Rad
Gel Doc 2000 system. The PFGE banding patterns of PNSP isolates
prepared by the standard protocol compared to the patterns of the same
strains prepared by the simplified protocol are shown in Fig.
1. The simplified protocol reliably
reproduces the banding pattern created with the standard protocol. Lane
9 (standard protocol) does have a faint band above 388 kbp which is not
seen in lane 8 (simplified protocol). This band most likely represents
unrestricted DNA. The PFGE patterns for the penicillin-susceptible
isolates prepared with the simplified protocol and those with the
standard protocol were also identical (data not shown).
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|
FIG. 1.
PFGE of invasive S. pneumoniae isolates.
Even-numbered lanes, simplified protocol; odd-numbered lanes, standard
protocol. Lane 1, lambda ladder; lanes 2 and 3, serotype 6A; lanes 4 and 5, serotype 9V; lanes 6 and 7, serotype 19F; lanes 8 and 9, serotype 23F (Spanish clone); lanes 10 and 11, serotype 19A; lanes 12 and 13, serotype 14; lanes 14 and 15, serotype 9V.
|
|
| |
DISCUSSION |
A wide variety of methods are available for the molecular
subtyping of infectious agents. In order to be useful, a
technique must provide adequate discrimination of
epidemiologically unrelated strains, result in gels with
high-resolution, reproducible banding patterns within a short
time; and be amenable to computer genetic relatedness analysis.
Unfortunately, most of the methods available are labor-intensive and
time-consuming.
PFGE has recently become an important method for determining microbial
genetic relatedness. While it provides excellent intraspecies discrimination among a broad array of bacterial and fungal pathogens, it is time-consuming.
Our simplified protocol offers substantial advantages over published
PFGE protocols for S. pneumoniae. By our method, any S. pneumoniae isolate can be optimally and efficiently
subtyped on a high-quality gel within 36 h, thereby eliminating a
key disadvantage of PFGE compared to BOX fingerprinting and restriction
fragment end labeling.
The elimination of the lysis step and deletion of all enzymes except
SmaI provides a simple, easy-to-use method for lysing S. pneumoniae. The lysis and digestion steps are
accomplished with a two-reagent alkaline buffer. We did note that a
small percentage of the organisms did not lyse during the first 6-h
incubation; however, when these isolates were incubated a second time
in the ES buffer overnight, adequate lysis did occur. The likelihood of
complete lysis increased if we incubated only one plug in each tube of
ES buffer. The need for endonuclease-free, filtered water is critical.
Nonfiltered water can contain significant concentrations of bacteria
and their accompanying RNA- and DNA-digesting enzymes. Without
proteinase K, these enzymes could cause degradation of the S. pneumoniae isolate's DNA. Moreover, residual proteinase K affects
the ability of the restriction enzyme to cut the DNA. The lack of
proteinase K may have improved our ability to restrict more of the
pneumococcal DNA, as the simplified protocol was not associated with
the faint high-molecular-weight band seen with the standard protocol
(Fig. 1, lane 9). The addition of a second block time on the CHEF DRIII
and the avoidance of heating of the DNA-containing fragments
reproducibly creates high-resolution banding patterns without any
background lane intensity. The lysis and digestion steps are
accomplished with a two-reagent alkaline buffer. S. pneumoniae undergoes autolysis by detergent activation of
N-acetylmuramyl-L-alanine amidase
(24). Perhaps N-lauroyl sarcosine, a detergent,
causes autolysis by activation of pneumolysin (18).
There are other rapid PFGE protocols for gram-positive (8, 15,
17) and gram-negative (7) bacteria.
Staphylococcus can be subtyped without proteinase K but
requires lysostaphin (8). Many gram-negative rods can be
subtyped without the lysis step but require digestion with proteinase K
(1). To the best of our knowledge, no other protocol has
been able to delete the lysis step and perform digestion without
proteinase K. Future studies could address whether additional organisms
could be subtyped with our simplified protocol. Moreover, this protocol
restricted the pneumococcal DNA with SmaI. Restriction of
pneumococcal DNA with additional enzymes, including ApaI,
could be performed in future investigations. While the time to gel
completion was diminished, the gels produced are often not of adequate
quality (15) and may not have the same banding pattern as
gels produced with standard protocols (17). In addition, the
other protocols require multiple buffers and/or enzymes due to the lack
of autolysins in most other bacteria (7, 15, 17). The unique
ability of S. pneumoniae to undergo autolysis allows
complete lysis and digestion without the addition of complex buffers or enzymes.
| |
ACKNOWLEDGMENTS |
We thank the participating hospital infection control
practitioners and microbiology laboratory personnel of the Maryland Emerging Infections Program for identifying the pneumococcal cases and
providing the bacterial isolates; Yvonne Dean-Hibbert and Lillian
Billmann for assistance in conducting surveillance; Kim Holmes for
assistance with data collection; and Althea Glenn for processing the
isolates. We gratefully acknowledge Alexander Tomasz for the capsular
type 23F Spanish-U.S. clone of Streptococcus pneumoniae. We
also thank Jan Patterson, Victor Yu, and Judy Johnson for their
assistance and guidance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Public
Health Infectious Diseases Laboratory, University of Pittsburgh
Graduate School of Public Health, A521 Crabtree Hall, 130 DeSoto
St., Pittsburgh, PA 15261. Phone: (412) 648-6401. Fax: (412) 648-6399. E-mail: McEllistremC{at}msx.dept-med.pitt.edu.
| |
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Journal of Clinical Microbiology, January 2000, p. 351-353, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
