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Journal of Clinical Microbiology, August 2000, p. 2914-2916, Vol. 38, No. 8
0095-1137/00/$04.00+0
Use of the Neisserial Lipoprotein (Lip) for
Subtyping Neisseria gonorrhoeae
David L.
Trees,*
Abbigail J.
Schultz, and
Joan
S.
Knapp
Division of AIDS, STD, and TB Laboratory
Research, National Center for Infectious Diseases, Centers for
Disease Control and Prevention, Atlanta, Georgia 30333
Received 17 March 2000/Returned for modification 25 April
2000/Accepted 25 May 2000
 |
ABSTRACT |
The pathogenic Neisseria species N. meningitidis and N. gonorrhoeae possess an outer
membrane lipoprotein, designated Lip, which is present in all strains
tested. The predicted protein sequence of Lip consists of a
consensus AAEAP amino acid repeat. The objective of this study was
to determine the feasibility of using the Lip repeat number and
sequence for subtyping of Neisseria gonorrhoeae. The
lip genes of each isolate were amplified by PCR and
sequenced to determine the repeat number and sequence. Among the 46 strains we examined, eight different Lip repeat numbers were
identified, with lengths of 11 (1 strain), 12 (14 strains), 13 (2 strains), 14 (10 strains), 15 (5 strains), 16 (10 strains), 17 (3 strains), and 20 (1 strain) repeats. Analysis indicated differences in the sequences within the repeats that resulted in
amino acid alterations in repeat classes that contained
multiple strains. Among the 46 isolates examined, we were able to
identify 17 unique Lip subtyping patterns.
| |
INTRODUCTION |
The pathogenic Neisseria
species Neisseria meningitidis and Neisseria
gonorrhoeae possess an outer membrane lipoprotein, designated Lip,
which is present in all strains tested. Characterization of purified
Lip has revealed that the protein has no A280;
glutamic acid, alanine, and proline account for 85% of the amino acids in the protein; no aromatic and sulfur-containing amino acids are
present; and Lip copurifies with lipid (2). The apparent molecular mass of Lip demonstrates strain variation, with a size range
of 18 to 30 kDa (3, 6). Three gonococcal lip
genes have been sequenced and have been shown to be highly homologous (1, 18). The predicted proteins from these sequences consist entirely of a consensus AAEAP amino acid pentamer repeat with the
predicted proteins varying between 13 and 19 repeats in length (1,
18). In this study, we have used Lip repeat number variation and
sequencing to develop a new gonococcal subtyping method which we can
use in combination which the other subtyping methods, such as
auxotype-serovar (A/S) classification, plasmid profile, TetM typing,
antimicrobial susceptibilities, and GyrA and ParC mutations, that we
currently use in our laboratory to increase our ability to
differentiate gonococcal strains in outbreak situations.
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MATERIALS AND METHODS |
Bacterial strains.
A total of 46 gonococcal isolates were
analyzed in this study: (i) 22 isolates from Cleveland, Ohio, isolated
in 1994; (ii) 12 isolates from Orange County, Calif., collected in
1995; (iii) 10 isolates from Hawaii collected from 1991 to 1994; and
(iv) 2 isolates that were an epidemiologically linked contact pair isolated in Hawaii in 1999. Isolates were provided to the Centers for
Disease Control and Prevention (CDC [Atlanta, Ga.]) frozen in
Trypticase soy broth containing 20% glycerol and were stored at
70°C.
Strain characterization and culture.
Isolates were
characterized by A/S as described previously (8, 10, 15).
Agar dilution susceptibilities of isolates to penicillin G,
tetracycline, spectinomycin, and ciprofloxacin were determined and
interpreted by National Committee for Clinical Laboratory
Standards-recommended methods, as described previously (11,
12). Isolates were assigned to penicillin-tetracycline resistance
phenotypes as described previously (7, 13, 14). The TetM
subtype was determined as previously described (16). Isolates were subcultured daily on GC base-IsoVitalex medium at 37°C
in a 5% CO2 atmosphere. Harvested cells were frozen in
Trypticase soy broth containing 20% glycerol and were stored at
70°C.
PCR analysis.
The lip genes were amplified by PCR
after extraction of the chromosomal DNA of each gonococcal strain. PCR
primers specific for lip, H8-1
(5'-CAAATTCAGCGATGAATTTCCAACCC 3') and H8-4
(5'-TATGAAGGTCAGGCATGTTTGTCGG 3'), were synthesized in the
National Center for Infectious Diseases Core Facility, CDC. PCR
amplification consisted of 35 cycles of denaturation at 94°C for
30 s, annealing at 64°C for 30 s, and extension at 72°C
for 60 s in reaction aliquots of 50 µl. Each PCR mixture
contained 5 µl of 10× PCR buffer (Boehringer Mannheim Biochemicals,
Indianapolis, Ind.), 1 µl of deoxynucleoside triphosphates (dNTP)
stock solution (containing 10 mM each dATP, dCTP, dGTP, and dTTP)
(Boehringer Mannheim), 0.5 µl of Taq DNA polymerase (5 U/µl) (Boehringer Mannheim), 2.5 µl of each of the appropriate primers (20 mM), 28.5 µl of sterile water, and 10 µl of template DNA. Ten microliters of PCR product from each isolate was visualized after electrophoresis on a 7.5% polyacrylamide gel, with size being
determined by comparison to DNA molecular weight marker VIII
(Boehringer Mannheim).
Sequencing analysis.
PCR products were purified for
sequencing with the High Pure PCR Product Purification kit (Boehringer
Mannheim). Sequencing reactions were performed in 20-µl volumes with
ABI Prism BigDYE Terminator Cycle Sequencing Ready Reaction kit
reagents (Applied Biosystems Division of Perkin-Elmer Corp., Foster
City, Calif.) containing 5.0 µl of Terminator Ready Reaction mix, 5 µl of PCR product template (approximately 100 ng), 3.2 µl of 20 µM primer, and 6.8 µl of sterile, deionized water. Reaction
mixtures were cycle sequenced on a GeneAmp PCR system 2400 (Perkin-Elmer) in 25 cycles of denaturation at 96°C for 10 s,
annealing at 50°C for 5 s, and extension at 60°C for 4 min.
Spin column purification with Centrisep Columns (Princeton Separations,
Adelphia, N.J.) was used to remove excess dye terminators and purify
reaction mixtures before electrophoresis. Electrophoresis of extension reaction mixtures was performed on the ABI Prism 377 DNA Sequencer (Applied Biosystems) with a 4% polyacrylamide gel (19:1
acrylamide-bisacrylamide; Bio-Rad Laboratories, Hercules, Calif.).
| |
RESULTS AND DISCUSSION |
Repeat variation and stability.
PCR analysis, with sequencing
used for confirmation, was used to determine the number of repeats
present in the Lip of gonococcal strains. Among the 46 strains
examined, eight different Lip repeat patterns were identified, with
lengths of 11 (1 strain), 12 (14 strains), 13 (2 strains), 14 (10 strains), 15 (5 strains), 16 (10 strains), 17 (3 strains), and 20 (1 strain) repeats (Fig. 1). To determine
the in vitro stability of the repeat lengths, two strains (one with 12 repeats and one with 20 repeats) were subcultured daily for 30 days,
with a sample taken and frozen each fifth day. Following the 30-day
passage, all samples were subjected to lip PCR and analyzed
on 7.5% acrylamide gels. Neither of the strains showed a change in the
length of the PCR product during the passage period; the Lip sequences
from day 1 and day 30 passages were identical for each strain (data not
shown). The in vivo stability of the Lip pattern was examined by
subtyping two epidemiologically linked gonococcal isolates which had
been collected in Hawaii during an outbreak investigation. Both
isolates possessed the 16b Lip pattern as well as having identical A/S class, plasmid profile, antimicrobial susceptibilities, and GyrA and
ParC mutations.
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FIG. 1.
Polyacrylamide gel showing the size of Lip PCR amplicons
versus the number of pentamer repeats present. The 465-bp and 591-bp
markers represent the sizes of PCR amplicons for 11 and 20 repeats,
respectively.
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Sequence analysis of similar repeat lengths.
DNA sequencing
data were examined to determine if the amino acid sequences were
identical for all Lips with the same number of repeats. Analysis
indicated differences in the sequences within the repeats that resulted
in amino acid alterations in each of the Lip repeat classes that
contained multiple strains. An example of the variation of the amino
acid sequence within a repeat number class is shown in Fig.
2 for the sequences observed in the
12-repeat class. In this class sequence, 12a differs from 12b by two
amino acids and 12a differs from 12c by eight amino acids. The other repeat classes exhibited similar sequence variation (data not shown).
The repeat class-sequence patterns, including the number of strains
with each pattern, are summarized in Table
1.
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FIG. 2.
Comparison of amino acid sequences of three variations
seen among strains with 12 pentamer repeats. Amino acid alterations
compared to sequence 12a are shown in lowercase italics for sequences
12b and 12c.
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TABLE 1.
Lip repeat-sequence subtyping patterns and number of
strains that exhibited each pattern among 46 isolates tested
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Use of Lip in subtyping.
To determine the feasibility of using
the Lip repeat-sequence pattern to subtype apparently related strains
of gonococci, we evaluated two subsets of strains which were
indistinguishable by A/S classification. One subset of nine strains
isolated in Orange County, Calif., all belonged to the Proto IB-3 A/S
class. Determination of antimicrobial resistance profiles divided the strains into three groups: three strains were penicillinase producing and tetracycline resistant (PP/TR), five strains were
tetracycline-resistant N. gonorrhoeae (TRNG), and one strain
was TRNG with chromosomal penicillin resistance. Subtyping of the TetM
determinant did not further differentiate the strains, in that all
strains contained the "Dutch-type" TetM gene (data not shown). Lip
repeat number-sequence analysis was able to identify the three PP/TR
strains as individual isolates with repeat-sequence patterns of 11a,
12c, and 15c (Table 2). Additionally, Lip
subtyping further divided the five TRNG strains, with three strains
having the 14a pattern, one strain having the 14c pattern, and one
having the 12c pattern (Table 2).
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TABLE 2.
A/S classification, MICs of penicillin and tetracycline,
and Lip subtypes of nine gonococcal strains isolated in Orange County,
Calif., between July and December 1995
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The second subset of 10 strains were isolated in Cleveland, Ohio, and
all belonged to the Pro IA-6 A/S class. Two strains
were TRNG, and
eight were classified as susceptible. As shown
in Table
3, both of the TRNG strains had a 15a Lip
pattern, while
the susceptible strains could be further subtyped with
Lip patterns
of 12a (four strains), 13a (one strain), and 14a (three
strains).
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TABLE 3.
A/S classification, penicillin and tetracycline MICs,
and Lip subtypes of 10 gonococcal strains isolated in
Cleveland, Ohio, in 1994
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|
In this study, we have evaluated the feasibility of using Lip repeat
number and amino acid sequence in the subtyping of
N. gonorrhoeae isolates. Of 46 isolates examined, we were able to
identify 17 unique Lip subtyping patterns which gave a discriminatory
index (
5) of 0.923 for this set of strains. The Lip
patterns,
both repeat number and sequence, of the two isolates tested
were
stable after 30 days of passage, and the same Lip pattern was
observed in an epidemiologically linked contact pair. Additionally,
by
determining the Lip pattern, we were able to differentiate
between
strains which had the same A/S class and antimicrobial
profile.
It should be noted, however, that Lip subtyping is not sufficiently
discriminatory to be used without additional gonococcal
subtyping
methods, such as A/S classification, TetM subtyping,
-lactamase
plasmid profiles, or GyrA-ParC mutation analysis in
fluoroquinolone-resistant isolates. An example of the need for
combination subtyping is the fact that seven isolates with the
14a Lip
pattern belonged to four different A/S classifications.
The need to use
multiple methods when subtyping gonococci exists,
even when one of the
methods is genomically based, such as pulsed-field
gel electrophoresis
(PFGE), discriminatory index of 0.997 (
17),
in that a report
by Marquez et al. (
9) identified multiple
A/S classes in a
single PFGE
pattern.
One significant advantage of Lip subtyping over TetM, plasmid, or
GyrA-ParC subtyping is that the Lip gene is present in all
gonococcal
strains and therefore does not require the presence
of antimicrobial
resistance. Presently, only A/S classification
and much more complex
methods such as PFGE can be used to subtype
susceptible gonococcal
isolates.
At this point, we envision Lip subtyping as being extremely useful in
situations such as those in Orange County and Cleveland
in which a
cluster of isolates belong to the same A/S class and
therefore may be
interpreted as an outbreak caused by a single
strain without the use of
an additional subtyping system. The
PCR stage of Lip subtyping will
quickly demonstrate if all isolates
contain the same number of repeats.
If a number of isolates have
the same repeat number, then the second
stage of sequencing can
be performed in an attempt to further
differentiate
isolates.
Sequences of all Lip types or sequences will be provided through the
internet (
4) to allow other investigators to compare
the Lip
patterns they observe with patterns previously reported.
We will
continue to add new patterns obtained during our studies
to the site
and will be willing to add any new patterns identified
by other
investigators.
| |
ACKNOWLEDGMENTS |
This study was supported in part by an appointment (A.J.S.) to
the Research Participation Program at the Centers for Disease Control
and Prevention administered by the Oak Ridge Institute for Science and Education.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Bacterial STD
Branch, Mailstop G-39, DASTLR, NCID, Centers for Disease Control and Prevention, Atlanta, GA 30333. Phone: (404) 639-2134. Fax: (404) 639-3976. E-mail: dlt1{at}cdc.gov.
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Journal of Clinical Microbiology, August 2000, p. 2914-2916, Vol. 38, No. 8
0095-1137/00/$04.00+0
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Abstract
Introduction
