1995, American Society for Microbiology. Differentiation of Neisseria ... tional Meeting of the International Society for STD Research,. Helsinki, Finland, 1993.).
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1995, p. 1039–1041 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 33, No. 4
Differentiation of Neisseria gonorrhoeae Isolates Requiring Proline, Citrulline, and Uracil by Plasmid Content, Serotyping, and Pulsed-Field Gel Electrophoresis LAI-KING NG,* MARIA CARBALLO,
AND
JO-ANNE R. DILLON†
National Laboratory for Sexually Transmitted Diseases, Laboratory Centre for Disease Control, Health Canada, Ottawa, Ontario, Canada K1A 0L2 Received 18 July 1994/Returned for modification 6 October 1994/Accepted 17 January 1995
A combination of DNA macrorestriction analysis using pulsed-field gel electrophoresis and a serotyping method using three panels of monoclonal antibody was used to discriminate 43 epidemiologically unrelated Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil (PCU2) into 35 groups. This indicates that PCU2 isolates of N. gonorrhoeae are not clonal. Neisseria gonorrhoeae isolates requiring proline, citrulline, and uracil (PCU2) (10) are phenotypically similar and thus difficult to differentiate by antibiogram, serotyping, and plasmid analysis (4, 5, 7, 12, 16). A previous evaluation of molecular or genetic typing methods such as restriction fragment length polymorphism (RFLP) analysis of rRNA genes (ribotyping) and isoenzyme typing methods provided less discrimination of PCU2 isolates than the Knapp serotyping (GSKnapp) method (12). However, ribotyping was useful as a subtyping method to further differentiate some isolates within a single serovar (12). In this study, we evaluate the ability of DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) and two monoclonal antibody serological typing schemes to discriminate between 43 epidemiologically unrelated N. gonorrhoeae PCU2 isolates selected from our culture collection (1979 to 1992) (Table 1). These isolates had been previously typed into nine serovars on the basis of the recommended Knapp scheme (9, 11). (These data were presented in part at the Tenth International Meeting of the International Society for STD Research, Helsinki, Finland, 1993.) The agarose plugs for PFGE were prepared as previously described by Taylor et al. (18) with the following modifications: 100 ml of cell suspension (1.5 3 109 CFU/ml) was prepared from overnight growth in supplemented GC medium base (GCMB; Difco, Detroit, Mich.), and the lysis solution contained 0.4 mg instead of 0.5 mg/ml of proteinase K. DNA from SpeI (10 U in 100 ml of buffer) digests were separated in a 1% LE agarose gel (Beckman Instruments, Inc., Palo Alto, Calif.). PFGE was carried out at 6 V/cm (200 V) for 20 h at 148C in 0.53 TBE buffer (18) with pulse times of 2 to 50 s and a linear ramp ratio of 1 by using CHEF.DRII [Biorad Laboratories (Canada) Ltd., Ontario]. Alternatively, the CHEFMAPPER (Biorad) was set at the automatic algorithm mode to separate fragment sizes of 20 to 500 kb. The 43 PCU2 isolates showed 15 PFGE patterns, PF-1 to
TABLE 1. Characterization of N. gonorrhoeae PCU2 isolates by serotyping and PFGE Type Isolate
NS8 NS787 NS724 NS263 WC41, WC59 NS697 C3, WC42, NS304, NS739, NS873 NS13 NS54 1-2152 NS384 NS571 NS97 NS576 NS791e 7537 8726e 3275 9017, 9014 2173 2324 7443, 8244 NS348 NS2194 NS211 NS1860 NS2123 8274 NS361 NS680 NS1754 NS1380 NS1862 3081, 2894, 1544
* Corresponding author. Present address: Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Symth Road, Health Sciences Centre, Ottawa, Ontario, Canada K1H 8M5. Phone: (613) 787-6777. Fax: (613) 738-5379. † Present address: Department of Microbiology and Immunology, Faculty of Medicine, Health Sciences Centre, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5.
a
GSKnappa
GSBygdemanb
IB-1
Babcejk
IB-2
Babcjk Bacejk
1039
Ph-seriesc
PFGE
RFLPd
PF-1 PF-11 PF-1 PF-7 PF-1 PF-6 PF-1
I ND I I I I I
PF-2 PF-3 PF-7 PF-5 Brpyu PF-10 Nontypeable PF-9 Nontypeable PF-1 Bropt PF-14 Bpyust PF-13 Bopt PF-14 Bpyut PF-8 PF-1 Bpt PF-12 Bust PF-7 Brpyust PF-7 Brpyu PF-15 Brpyust PF-1 Brpyu PF-11 PF-1 Bpyu PF-7 Bpyut PF-7 Brpyut PF-7 Bpyust PF-4 PF-7 PF-11 Bust PF-1 Bpust PF-1
I I II I III I ND IV ND ND ND ND ND ND ND III ND III ND ND ND I ND ND ND ND ND
Brpyut Bropst Brpst Brpyut Brpyust Brpyut
IB-3
Bacjk Babjk
IB-6
Baejk
IB-7
Baek Bak Bcegjk
IB-16
Bacej
IB-20 IB-25 IB-26
Baej Bbcej Babcegjk
Knapp et al., 1984 (11). Bygdeman et al., 1983 (2). c Sandstro ¨m et al., 1985 (15). d Ng and Dillon, 1993 (12); ND, not done. e Isolates with cryptic plasmid. b
Pattern
1040
NOTES
J. CLIN. MICROBIOL. TABLE 2. PFGE patterns: average molecular sizes of fragments Presence of PFGE patterna Fragment present 1
FIG. 1. Samples of different patterns of PFGE of SpeI fragments of genomic DNA of PCU2 isolates. Lane 1, l ladder with molecular size (in kilobases) indicated on the left margin. Lanes 2 to 16, PFGE types PF-1 to PF15, respectively. Lane 2, PF-1: NS8, NS724, WC41, WC59, C3, WC42, NS304, NS739, NS873, NS576, 9017, 9014, NS2194, NS1860, NS1862, 3081, 2896, 1544; lane 3, PF-2: NS13; lane 4, PF-3: NS54; lane 5, PF-4:NS680: lane 6, PF-5: NS384; lane 7, PF-6: NS697; lane 8, NS-7: NS263, 1-2152, 2324, 7443, 8244, NS2123, 8274, NS361, NS1754; lane 9, PF-8: 3275; lane 10, PF-9: NS97; lane 11, PF-10: NS571; lane 13 and 17, PF-11: NS211, NS787, NS1380; lane 13, PF-12:2173; lane 14, PF-13:7537; lane 15, PF-14: NS791, NS8726; lane 16, PF-15:NS384.
PF-15 (Fig. 1); all the patterns contained 8 common bands. The average molecular size (from at least three gels) of the bands is shown in Table 2. PFGE pattern PF-1 predominated, with 41.9% of the isolates belonging to this group and distributed among five GS-Knapp serovars (IB-1, IB-2, IB-6, IB-16, and IB-26). Serovar IB-2, the most predominant serovar of PCU2 isolates (5), comprised eight different PFGE patterns (Table 1). The next most common PFGE pattern was PF-7, which was found in 20.9% of the isolates belonging to eight of the nine GS-Knapp serovars (except IB-3) (Table 1). The three IB-3 isolates showed two unique PFGE patterns (Table 1), PF-13 and PF-14 (Fig. 1). The two monoclonal antibody serotyping systems used to subtype isolates comprising nine GS-Knapp serovars (9, 11) were the GS panel (GS-Bygdeman), with an additional four monoclonal antibodies (provided by Syva) using Bygdeman’s nomenclature (2), and the Pharmacia panel (Ph-panel) (Boule Diagnostics AB, Huddinge, Sweden) (15). Serovars were determined at least twice with each system. The GS-Bygdeman typing scheme resolved the 43 isolates into 13 serovars and further subtyped isolates belonging to GS-Knapp serovars IB-1 and IB-2 into four groups (Babcejk, Babcjk, Bacejk, and Bacjk), while serovar IB-6 isolates were subdivided into three groups (Baejk, Baek, and Bak) (Table 1). By using the Phpanel, 41 isolates were subtyped into 13 serovars and 2 isolates, NS97 (IB-2/Bacejk) and NS576 (IB-2/Bacjk), were untypeable (Table 1). Furthermore, in some instances, the Ph-panel was useful in distinguishing isolates that were similar with the GSKnapp/GS-Bydeman panel. For example, the 14 IB-2/Bacejk isolates could be further resolved into three different Ph serovars, with 9 out of 14 isolates belonging to serovar Brpyut. Using a combination of both the GS-panel and Ph-panel of monoclonal antibodies, the 43 isolates were differentiated into a total of 26 groups (Table 1). By combining serotyping and PFGE, the 43 isolates were
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
2
3
1 1 1
4
1 1
1
1 1 1 1 1 1 1 1 1 1 1 1 1
1
1
5
6
1
1 1
1 1 1 1 1 1 1
8
9 10 11 12 13 14 15
1 1 1
1 1 1
1
1
1
1
1 1
1 1 1
1 1 1
1
1 1 1 1 1 1* 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1
7
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1
Fragment size (kb)
643.3 471.3 430.0 408.2 371.7 364.0 277.4 262.5 257.8 240.2 227.9 217.4 210.0 200.2 191.0 180.8 168.0 159.9 154.3 145.4 141.4 134.0 127.0 111.4 98.2 84.4 76.8 60.0 45.6 40.6 33.4 28.8
a PF-1: NS8, NS724, WC41, WC59, C3, WC42, NS304, NS739, NS873, NS576, 9017, 9014, NS2194, NS1860, NS1862, 3081, 2896, 1544; PF2: NS13; PF-3: NS54; PF-4:NS680: PF-5: NS384; PF-6: NS697; NS-7: NS263, 1-2152, 2324, 7443, 8244, NS2123, 8274, NS361, NS1754; PF-8: 3275; PF-9: NS97; PF-10: NS571; PF-11: NS211, NS787, NS1380; PF-12:2173; PF-13:7537; PF-14: NS791, NS8726; PF-15: NS384. 1, presence of specific band in PFGE profile. *, dimer.
distinguished into 35 groups, further differentiating 9 isolates belonging to the predominant serovar IB-2/Bacejk/Brpyut into five PFGE types (Table 1). Isolates not further distinguished by PFGE belonged to serovar IB-6/Baejk/Bpyut, IB-7/Bcegjk/ Brpyust, and IB-26/Babcegjk/Bpust (Table 1). Previously, most PCU2 isolates were shown to be plasmid free except for isolate NS791 (serovar IB-3), which it also had a unique ribotype and multilocus locus enzyme electrophoresis pattern (12). In this study, plasmid analysis (6) showed that isolates 8726 and 9684 (serovar IB-3) carried the same 4.3-kb variant cryptic plasmid as NS791. The restriction endonuclease digestion patterns of plasmids of isolates 8726 and 9684 were similar to that of pNS791, which has a unique HindIII site (data not shown). All three 4.3-kb plasmids had identical MspI (Fig. 2, lanes 3 to 5) and HinfI (Fig. 2, lanes 7 to 9) fragments which differed from the reference 4.2-kb cryptic plasmid pLCDC1 (Fig. 2, lanes 2 and 6) in the smallest fragments (Fig. 2). Previously pNS791 was shown to contain an insertion of approximately 0.14 kb in the 0.46-kb HinfI/MspI fragment of pLCDC1 (12). Only 3 of the 5,538 PCU2 isolates collected from March 1988 to March 1993 were serovar IB-3, and all contained plasmids, possibly indicating that this unusual serovar may be able to acquire the gonococcal cryptic plasmid. Both isolates NS791 and 8726 had the same PFGE pattern
VOL. 33, 1995
NOTES
1041
For clonal analysis, the choice of methods or target genetic loci will affect the success of detecting genomic diversity (13). We found that ribotyping based on rRNA genes is less sensitive than either monoclonal antibody serotyping or macrorestriction endonuclease analysis by PFGE. In contrast to our previous results using ribotyping to discriminate between PCU2 isolates (12), genomic variations were observed within PCU2 serovars by using PFGE, including serovar IB-2. We thank Diane Taylor, Department of Microbiology and Infectious Diseases, University of Alberta, Edmonton, Alberta, Canada, for her advice on the preparation of agarose plugs. This project was funded in part by the National Biotechnology Strategy funding to Federal Government laboratories and the Canadian Bacterial Diseases Network. REFERENCES
FIG. 2. Restriction endonuclease analysis of cryptic plasmids in PCU2 and reference (nonrequiring, LCDC1) isolates. Lanes 1 and 10, 1-kb molecular size markers (BRL) (top to bottom: 12,216, 11,198, 10,180, 9,162, 8,144, 7,126, 6,108, 5,090, 4,072, 3,054, 2,036, 1,636, 1,018 bp). Lanes 2 to 5, MspI digests of plasmids from LCDC1, NS791, 8276, and 9684, respectively. Lanes 6 to 9, HinfI digests of plasmids LCDC1, NS791, 8276, and 9684, respectively.
(PF-13) but could be distinguished with the Ph-panel of monoclonal antibodies (Table 1). In previously published maps of N. gonorrhoeae MS11-N198 (1) and FA1090 (3), which are not PCU2 isolates, different SpeI patterns were reported and both patterns, based on reported fragment sizes, appeared different from the profiles obtained in this study. A recent study (14) observed 38 SpeI PFGE patterns from 48 gonococcal isolates comprising 18 auxotype/serovar classes other than PCU2. The PFGE method requires standardization for routine analysis; at present, most typing studies do not report fragment sizes of different PFGE patterns and there are no established reference gonococcal strains for standardization. As a subtyping method, serotyping is more simple and rapid than PFGE. PFGE has the advantage that all isolates are typeable while some isolates (two in this study identified with the Ph-panel) may not be typeable by serotyping. Both methods were superior than ribotyping, which in turn was more discriminatory than multilocus enzyme electrophoresis (12). A combination of methods may be necessary for subtyping isolates which appear clonal by classical methods, in order to achieve the desirable degree of discrimination required for these isolates. In general, the combination of auxotyping, serotyping, and plasmid profile analysis are adequate for discriminating between most N. gonorrhoeae isolates (8). Therefore, PFGE, ribotyping, or extended serotyping with additional monoclonal antibodies may be not advisable because the data obtained may be too complex for clinical analysis.
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