A multiplex polymerase chain reaction to differentiate ß-lactamase plasmids of Neisseria gonorrhoeae

H. M. Palmera,*, J. P. Leemingb and A. Turnera

a Genitourinary Infections Reference Laboratory, Public Health Laboratory, Bristol Royal Infirmary, Bristol BS2 8HW; b Public Health Laboratory, Bristol Royal Infirmary, Bristol BS2 8HW, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In penicillinase-producing Neisseria gonorrhoeae (PPNG), resistance to penicillin may be mediated by one of several related plasmids of different sizes. These include the Asian, African and Rio/Toronto plasmids. Identification of these plasmids provides useful epidemiological information, but has necessitated plasmid purification and gel analysis. We have developed a rapid, simple multiplex polymerase chain reaction (PCR) which discriminates between the ß-lactamase resistance plasmids that are frequently found in strains of N. gonorrhoeae. Amplicons of 1191, 958 and 650 bp were produced from strains containing the African, Asian and Rio/Toronto plasmids, respectively, whilst no products resulted from non-PPNG strains harbouring the cryptic, conjugative or tetracycline resistance plasmids. PCR analysis of 123 strains of PPNG identified 60 strains with African, 16 strains with Asian and 47 strains with Rio/Toronto plasmids and showed complete agreement with the standard plasmid analysis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In penicillinase-producing Neisseria gonorrhoeae (PPNG) penicillin resistance is mediated by the TEM-1 ß-lactamase gene, which is carried on several related plasmids. Strains carrying the Asian plasmid (reported size range 4.4–4.7 MDa) were first isolated in the USA in 19761 and named according to their epidemiological origin in the Far East. Comparative molecular analysis has suggested that the Asian plasmid is probably the ancestral plasmid from which other deletion or insertion derivatives have been generated.2 Strains carrying the African plasmid (reported size range 3.2–3.4 MDa) were isolated in the UK in 1976.3,4 The African plasmid has a 2.1 kb deletion when compared with the Asian plasmid. Two other deletion derivatives were detected in 1984 by separate research groups and designated the Toronto (3.05 MDa)5 and Rio (2.9 MDa)6 plasmids. Due to the similarity in size of these two plasmids, they are not differentiated in most epidemiological surveys and all plasmids of 2.9–3.05 MDa are reported by some authors as Toronto710 and by others as Rio plasmids.11 For the purposes of this paper, Rio and Toronto plasmids will be referred to as 3.0 MDa plasmids when mentioned collectively. Strains harbouring Asian, African or 3.0 MDa plasmids occur throughout the world and in 1998 PPNG accounted for 5.5% (111/2028) of gonococcus laboratory reports to the Communicable Disease Surveillance Centre (I. Simms, personal communication).

Other plasmids have been reported, including the Nimes plasmid (4.0 MDa),12 the New Zealand plasmid (6.0 MDa)13 and a single isolate carrying an unnamed variant (3.9 MDa) isolated in the Phillipines,14 but global dissemination of these plasmids has not followed their emergence as yet.

Analysis of the plasmids provides useful epidemiological data, but involves laborious plasmid purification, typically by the alkaline lysis method. We have developed a rapid, simple polymerase chain reaction (PCR)-based method to differentiate between the ß-lactamase-producing plasmids occurring commonly in N. gonorrhoeae.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

One hundred and fifty three isolates of N. gonorrhoeae referred to the Genitourinary Infections Reference Laboratory (GUIRL) or from clinical specimens submitted to the laboratory by primary care physicians in Bristol were used in this study. These comprised 123 PPNG, including 10 local isolates, 28 non-penicillinase-producing tetracycline-resistant N. gonorrhoeae (TRNG), including two local isolates, and two referred isolates which were non-PPNG, non-TRNG. Also, five strains held in the culture collection of the GUIRL were used as controls and included in each batch of isolates analysed. The control strains were SB5 (containing the Asian, the cryptic and the conjugative plasmids), GR4103 (containing the African, cryptic and conjugative plasmids), GR12894 (containing the 3.0 MDa, cryptic and conjugative plasmids), TR01 (containing the tetracycline resistance and cryptic plasmids; kindly provided by Dr C. A. Ison) and the World Health Organization control strain B (containing the cryptic plasmid). The control strains were also used to determine the sensitivity of the PCR.

Isolates of N. gonorrhoeae were grown on heated blood agar plates incubated at 37°C with 5% CO2 overnight. ß-Lactamase activity was determined by the method of Hodge et al.,15 using the Oxford strain of Staphylococcus aureus (NCTC 6571). Those gonococcal strains with satellite growth of S. aureus on a heated blood agar plate containing 1 mg/L ampicillin were considered to be PPNG and were selected for further analysis by both plasmid isolation and PCR methods. Isolates that demonstrated resistance to tetracycline by disc diffusion (10 µg disc) were also included in the plasmid analysis.

Plasmid DNA preparation and electrophoresis

The overnight growth of N. gonorrhoeae from one or two heated blood agar plates (containing 1 mg/L ampicillin for PPNG strains) was harvested and the plasmid DNA extracted by an alkaline lysis method.16 The purified plasmid DNA (18 µL) was analysed by gel electrophoresis with a 1% agarose gel, stained with ethidium bromide (10 mg/L) and visualized using UV transillumination.

Preparation of DNA for PCR

A 1 µL loopful of bacteria was suspended in 150 µL 5% Chelex-100 resin (Bio-Rad, Hemel Hempstead, UK) slurry in distilled water. Samples were heated (95°C for 10 min), centrifuged (2 min at 13 000g) and 5 µL of the supernatant used for PCR either immediately or after storage at –20°C.

PCR

Primers were designed using representative sequences of the African, Asian, Toronto and Rio plasmids that have been deposited in GenBank (accession numbers U20374, U20375, U20419 and U55934). Primers were positioned either side of the published deletion sites, avoiding regions of repeated sequence and the TEM-1 gene. Details of the plasmids, primers and predicted product sizes are summarized in Tables I and IIGoGo.


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Table I. Primers used for the detection of the ß-lactamase-producing plasmids of N. gonorrhoeae and the confirmation of their identity by DNA sequencing
 

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Table II. Predicted sizes of deletions and PCR products from the ß-lactamase-producing plasmids of N. gonorrhoeae
 
Target DNA (5 µL) was added to a 50 µL reaction containing 1 x ammonium sulphate-based PCR buffer (Qiagen, Crawley, UK), 1.5 mM MgCl2, dNTPs (20 µM or 200 µM), 1.25 U Taq DNA polymerase (Qiagen) and 0.5 µM of each primer, and overlaid with mineral oil. A hot start was achieved manually, by adding the target DNA to the reaction mixture at 75°C. Aliquots of ready-to-use reaction mixtures were also frozen at –20°C. These were heated to 75°C for 5 min before the addition of target DNA. PCR was performed using an Omnigene thermal cycler (Hybaid, Middlesex, UK) with the following cycle: 94°C, 3 min, then 35 cycles of 94°C for 15 s, 64°C for 15 s, 72°C for 1 min. PCR products (20 µL) were analysed by gel electrophoresis with a 0.7% agarose gel and stained and visualized as described above.

To confirm the identity of the PCR products, those from the control strains GR4103, SB5 and GR12894 were fully sequenced in both directions (additional primers were required to sequence the products of GR4103 and SB5; see Table IGo). In addition, all PCR products were digested with an appropriate restriction enzyme (AluI for the products of Asian and African plasmids and HaeIII for the product of the 3.0 MDa plasmid) by standard methods. Digested products were analysed by gel electrophoresis with a 1.2% agarose gel.

Assessment of sensitivity of the PCR

Preparations of whole cell DNA and purified plasmid DNA were made from the SB5, GR4103 and GR12894 control strains carrying the Asian, African and 3.0 MDa plasmids, respectively. Whole cell DNA was prepared as previously described17 and plasmid DNA was prepared as described above except that 10 mg/L RNase was added during the lysis procedure. DNA preparations were quantified spectrophotometrically at 260 nm using GeneQuant (Amersham Pharmacia Biotech Ltd, Amersham, UK). In addition, suspensions of whole bacteria from each of the control strains were made in water to an OD540 = 1.0 (approximately 108 cfu/mL). Serial dilutions of each DNA and bacterial suspension were made and PCR carried out to assess the sensitivity of the assay.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amplicons of characteristic size were produced for the African plasmid (1191 bp) from strain GR4103, the Asian plasmid (958 bp) from strain SB5 and the 3.0 MDa plasmid (650 bp) from strain GR12894. No products were obtained from strain B (containing the cryptic plasmid) or strain TR01 (containing the cryptic and the tetracycline resistance plasmids). Sequencing confirmed the identity of the products from the control strains (>99% identity with the relevant regions of the Asian plasmid, GenBank accession number U20374). Restriction digestion of amplicons from the control strains resulted in fragments of the predicted sizes: African plasmid, 896 bp + 295 bp; Asian plasmid, 663 bp + 295 bp; and 3.0 Mda plasmid, 475 bp + 175 bp (FigureGo).



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Figure. Agarose gel of PCR products before (a) and after (b) restriction digestion. Lanes 1 and 20, 100 bp ladder (Life Technologies, Paisley, UK); lane 2, WHO control strain B containing the cryptic plasmid; lane 3, GR12894 containing the cryptic, 3.0 MDa and conjugative plasmids; lane 4, GR4103 containing the African, cryptic and conjugative plasmids; lane 5, SB5 containing the Asian, the cryptic and the conjugative plasmids; lane 6, TR01 containing the tetracycline resistance and cryptic plasmids; lanes 7–19, examples of PPNG isolates containing the African (lanes 7, 10–13 and 15–17), Asian (lanes 8, 14 and 18) and 3.0 MDa plasmids (lanes 9 and 19).

 
With strains SB5, GR4103 and GR12894 the detection limit was 1 ng total DNA, 0.1 ng purified plasmid and c. 100 whole bacterial cells. This sensitivity was achieved using 200 µM dNTPs. For routine analysis, where the amount of target DNA is not limiting, it was found that 20 µM dNTPs was adequate and this was adopted as the standard. No differences in amplification were seen when using freshly prepared PCR mixes or aliquots that were frozen for 3 months.

With the standardized PCR conditions, analysis of 123 PPNG strains from widely different geographical origins identified 60 African, 16 Asian and 47 3.0 MDa plasmids (Table IIIGo). The identity of all PCR products was confirmed by restriction digestion. These data showed complete agreement with the conventional plasmid analysis. All 30 non-PPNG strains tested were negative by PCR.


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Table III. The plasmid types detected in 123 PPNG and their epidemiological origins
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Lactamase-producing plasmids of N. gonorrhoeae have been detected by hybridization18 and by PCR.19 However, neither method differentiated the different molecular weight TEM-1-encoding plasmids that occur in this species, so they are of limited value in the epidemiological surveillance of PPNG. Here we present the first multiplex PCR method that differentiates between the African, Asian and 3.0 MDa plasmids of N. gonorrhoeae. Each plasmid type has been detected in several isolates that are unrelated by virtue of their disparate geographical origins, demonstrating the robustness of the method. As expected, the majority (9/13) of PPNG from Asian countries harboured the Asian plasmid type. Most PPNG known to have been acquired within the UK contained the African plasmid type.

Amplicons of indistinguishable size were produced from all isolates bearing 3.0 MDa plasmids. Although 3.05 MDa (Toronto) and 2.9 MDa (Rio) ß-lactamase-encoding plasmids were described separately in 1984, these types were subsequently compared by restriction analysis and found to be indistinguishable.2 Furthermore, sequence data deposited in GenBank suggests that the site of deletion is the same in each (accession numbers U20419 and U55934), but different from that of the African plasmid. It is probable that only one 3.0 MDa plasmid type exists, but that simultaneous reporting of the emergence of this plasmid has resulted in two independent accounts by separate research groups, each assigning the plasmid a different name. The difference in epidemiological origin does not controvert this possibility, since international travel could easily account for the presence of related strains in widely different geographical areas. The strains containing the original Rio and Toronto plasmids were not available to the authors but the observation that all 47 isolates bearing the 3.0 MDa plasmid gave 650 bp PCR products is consistent with this conclusion.

The simplicity of the PCR method is an important feature if it is to be adopted for typing gonococcal plasmids on a routine basis. When compared with traditional plasmid purification, the PCR is technically undemanding, requires much less hands-on preparation time (30 min versus 2 h for 10–20 samples), and very little sample material (a 1 µL loopful of bacteria versus the growth from one or two agar plates). In addition, for ease of use and quality assurance, PCR mixes can be made in large batches, aliquoted and stored frozen for several months with no observable difference in performance. For both plasmid purification and PCR, the analysis is dependent on subsequent agarose gel electrophoresis. However, the analysis of short linear amplicons is less exacting than that of large circular plasmids: the latter may assume various different conformations and interpretation of the data requires some skill. Analysis of tetracycline resistance plasmids is often performed concurrently and this may be achieved by PCR using one of the three published tetM PCR protocols.2022 We have found that the thermal cycle presented here, with the annealing temperature adjusted to 58°C, is suitable for both this assay and the tetM PCR recently published by Turner et al.22

This PCR is intended as a method for characterizing pure cultures of N. gonorrhoeae and for this purpose it is both sensitive and specific. In circumstances where non-culture diagnosis of N. gonorrhoeae is advantageous, non-culture-based typing methods for antibiotic resistance determination will also be valuable. We have shown that nanogram amounts of purified DNA and c. 100 bacteria can be detected, therefore this method would probably be sufficiently sensitive for this purpose. However, the specificity of the test would require further investigation. ß-Lactamase plasmids are occasionally found in strains of N. meningitidis23 and other closely related plasmid types occur in Haemophilus ducreyi, Haemophilus influenzae and Haemophilus parainfluenzae.24 The possibility that these plasmids might be detected has not been investigated.

In summary, this is the first reported multiplex PCR that differentiates between different gonococcal ß-lactamase-producing plasmids. It is simple and reproducible and offers a significant methodological advance for the epidemiological surveillance of PPNG. Further evaluation may also establish a role for the assay in enhancing nonculture investigation of gonococcal infection.


    Acknowledgments
 
The authors thank Mrs K. Gough and Ms M. Hemming for excellent technical assistance, and Dr A. Herring for critical review of this manuscript.


    Notes
 
* Corresponding author. Tel: +44-117-9282557; Fax: +44-117-0200162; E-mail: Helen.Palmer{at}ubht.swest.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ashford, W. A., Golash, R. G. & Hemming, V. G. (1976). Penicillinase-producing Neisseria gonorrhoeae. Lancet ii, 657–8.

2 . Dillon, J. A. & Yeung, K. H. (1989). Beta-lactamase plasmids and chromosomally mediated antibiotic resistance in pathogenic Neisseria species. Clinical Microbiology Reviews 2 Suppl., S125–33.[ISI][Medline]

3 . Percival, A., Rowlands, J., Corkill, J. E., Alergant, C. D., Arya, O. P., Rees, E. et al. (1976). Penicillinase-producing Gonococci in Liverpool. Lancet ii, 1379–82.

4 . Phillips, I. (1976). Beta-lactamase-producing, penicillin-resistant gonococcus. Lancet ii, 656–7.

5 . Yeung, K. H., Dillon, J. R., Pauze, M. & Wallace, E. (1986). A novel 4.9-kilobase plasmid associated with an outbreak of penicillinase-producing Neisseria gonorrhoeae. Journal of Infectious Diseases 153, 1162–5.[ISI][Medline]

6 . van Embden, J. D., Desses-Kroon, M. & van Klingeren, B. (1985). New ß-lactamase plasmid in Neisseria gonorrhoeae. Journal of Antimicrobial Chemotherapy 15, 247–50.[ISI][Medline]

7 . Harnett, N., Brown, S., Terro, R., Krishnan, C., Pauze, M. & Yeung, K. H. (1997). High-level tetracycline-resistant Neisseria gonorrhoeae in Ontario, Canada—investigation of a cluster of isolates, showing chromosomally mediated resistance to penicillin combined with plasmid-mediated resistance to tetracycline. Journal of Infectious Diseases 176, 1269–76.[ISI][Medline]

8 . Sarafian, S. K., Rice, R. J., Ohye, R. G., Higa, H. & Knapp, J. S. (1994). Diversity of isolates of penicillinase-producing Neisseria gonorrhoeae (PPNG) in Honolulu, Hawaii: 1982–1991. Sexually Transmitted Diseases 21, 332–7.[ISI][Medline]

9 . Reimann, K., Bollerup, A. C. & Lind, I. (1992). The emergence of penicillinase-producing Neisseria gonorrhoeae strains carrying the 4.9 kb (Toronto) plasmid in Denmark and of a novel large plasmid in two non-penicillinase-producing Neisseria gonorrhoeae strains. Sexually Transmitted Diseases 19, 206–12.[ISI][Medline]

10 . Chu, M. L., Ho, L. J., Lin, H. C. & Wu, Y. C. (1992). Epidemiology of penicillin-resistant Neisseria gonorrhoeae isolated in Taiwan, 1960–1990. Clinical Infectious Diseases 14, 450–7.[ISI][Medline]

11 . Vaz Pato, M. V., Ribeiro Pires I., Van Klingeren, B., Louro, D. & Rosa, H. (1988). Penicillinase-producing Neisseria gonorrhoeae isolated in Lisbon 1982–1987. Pathologie Biologie 36, 639–42.[ISI][Medline]

12 . Gouby, A., Bourg, G. & Ramuz, M. (1986). Previously undescribed 6.6-kilobase R plasmid in penicillinase-producing Neisseria gonorrhoeae. Antimicrobial Agents and Chemotherapy 29, 1095–7.[ISI][Medline]

13 . Brett, M. (1989). A novel gonococcal ß-lactamase plasmid. Journal of Antimicrobial Chemotherapy 23, 653–4.[ISI][Medline]

14 . Knapp, J. S., Mesola, V. P., Neal, S. W., Wi, T. E., Tuazon, C., Manalastas, R. et al. (1997). Molecular epidemiology, in 1994, of Neisseria gonorrhoeae in Manila and Cebu City, Republic of the Philippines. Sexually Transmitted Diseases 24, 2–7.[ISI][Medline]

15 . Hodge, W., Ciak, J. & Tramont, E. C. (1978). Simple method for detection of penicillinase-producing Neisseria gonorrhoeae. Journal of Clinical Microbiology 7, 102–3.[ISI][Medline]

16 . Bennett, P. M., Heritage, J. & Hawkey, P. M. (1986). An ultra-rapid method for the study of antibiotic resistance plasmids. Journal of Antimicrobial Chemotherapy 18, 421–4.[Abstract]

17 . Nassif, X., Puaoi, D. & So, M. (1991). Transposition of Tn1545-delta 3 in the pathogenic Neisseriae: a genetic tool for mutagenesis. Journal of Bacteriology 173, 2147–54.[ISI][Medline]

18 . Sanchez-Pescador, R., Stempien, M. S. & Urdea, M. S. (1988). Rapid chemiluminescent nucleic acid assays for detection of TEM-1 ß-lactamase-mediated penicillin resistance in Neisseria gonorrhoeae and other bacteria. Journal of Clinical Microbiology 26, 1934–8.[ISI][Medline]

19 . Simard, J.-L. & Roy, P. H. (1993). PCR detection of penicillinase-producing Neisseria gonorrhoeae. In Diagnostic Molecular Microbiology: Principles and Applications, (Persing, D. H., Smith, T. F., Tenover, F. C. & White, T. J., Eds), pp. 543–6. American Society for Microbiology, Washington, DC.

20 . Ison, C. A., Tekki, N. & Gill, M. J. (1993). Detection of the tetM determinant in Neisseria gonorrhoeae. Sexually Transmitted Diseases 20, 329–333.[ISI][Medline]

21 . Xia, M., Pang, Y. & Roberts, M. C. (1995). Detection of two groups of 25.2 MDa Tet M plasmids by polymerase chain reaction of the downstream region. Molecular and Cellular Probes 9, 327–32.[ISI][Medline]

22 . Turner, A., Gough, K. R. & Leeming, J. P. (1999). Molecular epidemiology of tetM genes in Neisseria gonorrhoeae. Sexually Transmitted Infections 75, 60–6.[Abstract]

23 . Bäckman, A., Orvelid, P., Vazquez, J. A., Sköld, O. & Olcén, P. (1998). Characterization including sequence determination of ß-lactamase genes in two Neisseria meningitidis plasmids. In Eleventh International Pathogenic Neisseria Conference, Nice, France, 1998. (Nassif, X., Quentin-Millet, M.-J. & Taha, M.-K., Eds), Abstract 43, p. 119. EDK, Paris.

24 . Brunton, J. L., Clare, D., Ehrman, N. & Meier, M. A. (1983). Evolution of antibiotic resistance plasmids in Neisseria gonorrhoeae and Haemophilus species. Clinical and Investigative Medicine—Medecine Clinique et Experimentale 6, 221–8.

Received 8 April 1999; returned 14 September 1999; revised 25 October 1999; accepted 16 January 2000





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