Chloramphenicol-resistant Neisseria meningitidis containing catP isolated in Australia

Tiffany R. Shultz1, John W. Tapsall1,*, Peter A. White2, Catherine S. Ryan3, Dena Lyras3, Julian I. Rood3, Enzo Binotto4 and Christopher J. L. Richardson5

1 Department of Microbiology, South Eastern Sydney Area Health Services, The Prince of Wales Hospital, Sydney 2031; 2 School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney 2052; 3 Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University 3800; 4 Department of Microbiology and Infectious Diseases, South West Area Pathology Service, Locked Bag 7090 Liverpool BC 1871, Australia; 5 Department of Microbiology, Women’s & Children’s Health Services (WA); Princess Margaret Hospital, GPO Box D184, Perth, Western Australia 6001

Received 9 June 2003; returned 9 July 2003; revised 19 August 2003; accepted 22 August 2003


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Earlier workers have described chloramphenicol resistance in meningococci isolated from cerebrospinal fluid sampled in patients in Vietnam (11 cases) and France (one case) during 1987–1996. Here we describe two distinct serogroup B strains isolated in Australia in 1994 and 1997, and found among ~1400 invasive meningococcal isolates examined in Australia over a 9 year period. Both were phenotypically chloramphenicol resistant on disc, Etest and agar inclusion MIC and acetylated chloramphenicol examination. DNA amplification and sequencing confirmed the presence of catP and the 3' end of tnpV from Tn4451, a mobilizable element from Clostridium perfringens, although other sequences were not present. Tn4451 has inserted into a gene designated TIGR locus NMB1350 in both isolates with no loss of DNA and no apparent interruption of virulence genes. This second report of chloramphenicol-resistant meningococci is in a setting with a very low volume of systemic chloramphenicol use, but where the high topical use may contribute to recombination events in vivo. Methods for screening for chloramphenicol resistance in meningococci and the in vitro parameters that define this resistance are ill defined.

Keywords: N. meningitidis, chloramphenicol resistance, antibiotic use, chloramphenicol acetyltransferase, transposons


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Chloramphenicol has been used extensively to treat bacterial infections in many countries for many years. In Australia, parenteral use of chloramphenicol is now uncommon, although topical therapies, including eye preparations, are still widely employed. In less developed settings, chloramphenicol retains a major role in the treatment of bacterial meningitis, including that resulting from Neisseria meningitidis.

Despite this widespread use, chloramphenicol resistance in meningococci is rarely reported. Galimand et al.1 described 12 instances of high-level chloramphenicol resistance in meningococci isolated from cerebrospinal fluid sampled in patients in Vietnam (11 cases) and France (one case) during 1987–1996. The resistance was due to the action of the enzyme, chloramphenicol acetyltransferase (CAT), which mediates O-acetylation of chloramphenicol, destroying its affinity for bacterial ribosomes and thus its ability to inhibit bacterial growth. There are two separate families of CAT enzymes in bacteria, CATA and CATB.2 All CAT enzymes are able to acetylate chloramphenicol to form 3-acetoxy-chloramphenicol. However, 1-acetoxy-chloramphenicol, formed by a non-enzymatic rearrangement of 3-acetoxy-chloramphenicol, is only acetylated by CATA enzymes.2 The cat genes encoding production of CAT enzymes are often found in mobile genetic elements, such as plasmids, transposons or integrons. By hybridization, PCR and sequencing techniques, Galimand et al.1 found that in each of their 12 isolates a catP gene was present. The catP gene encodes a member of the CATA family of enzymes and is found on the integrative mobilizable Tn4451 from Clostridium perfringens.1 It is therefore thought that the catP gene in these neisserial isolates may have been derived from C. perfringens.3 However, direct integration of Tn4451 was unlikely since most of the surrounding genes of Tn4451 had been deleted. The meningococci involved were all of serogroup B, a highly transformable meningococcal group.

There have been few systematic studies on the prevalence of chloramphenicol-resistant meningococci (CmRNm). Interest in developed countries, where the drug is used infrequently, is low, and facilities for obtaining isolates for testing in settings with high caseloads of meningococcal meningitis are fragmentary. Furthermore, methods for screening for CmRNm are not well described and criteria for defining the phenomenon are unclear. No CmRNm were detected in studies by Nicolas et al.4 (24 serogroup A strains from Africa), Blondeau & Yaschuk5 (78 serogroup B, 38 serogroup C and five serogroup Y strains from Canada), Pascual et al.6 (34 serogroup B and 18 serogroup C strains from Spain) and Tondella et al.7 (33 serogroup A strains from Africa). The methods for determination of chloramphenicol resistance included broth microdilution and Etest MICs and in one instance detection of the catP gene.7

We report here the characterization of two further isolates of CAT-mediated CmRNm from invasive disease, where the resistance was conferred by the presence of a catP gene. The isolates were detected in an optional component of the programme of antimicrobial resistance surveillance conducted by the National Neisseria Network of Australia (NNN).


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Clinical cases

Case 1. The patient arrived in Australia from Vietnam in 1992 aged 5 years. In 1994 when aged 7 years, she presented as a clinical case of meningitis, and N. meningitidis serogroup B was grown from blood cultures. She was treated with intravenous penicillin and cefotaxime, and recovered. Rifampicin was given orally for 3 days. There was no history of chloramphenicol exposure at any time, although she was given isoniazid for 12 months in 1992 when she gave a positive Mantoux reaction, but with a normal chest X-ray.

Case 2. In 1997, a 78-year-old Caucasian woman was found at home moribund, and died shortly after admission to hospital. N. meningitidis serogroup B was isolated from her blood cultures. No history of prior antibiotic exposure was obtained.

Laboratory methods

The NNN is a continuing collaborative programme of surveillance of isolates of N. meningitidis from invasive cases of meningococcal disease conducted by reference laboratories in each State and Territory in Australia. The programme includes surveillance of the distribution of MICs of selected antibiotics including penicillin, ceftriaxone, rifampicin and ciprofloxacin. Chloramphenicol susceptibility is not determined routinely as it is no longer used for systemic treatment of meningococcal disease in Australia. Individual members of the NNN, however, conduct surveillance on other antibiotics including chloramphenicol. All isolates were confirmed as N. meningitidis on the basis of standard laboratory tests and serogrouped, serotyped and serosubtyped with standard antisera (National Institute for Public Health and the Environment—RIVM, Bilthoven, The Netherlands) as part of standard NNN examinations. Chloramphenicol resistance was demonstrated in the coordinating laboratory by the calibrated dichotomous sensitivity test method,8 and Etest MIC determinations (AB Biodisc, Sweden) on chocolate agar using Columbia agar (Oxoid, Basingstoke, UK) as the basal medium, and by the agar plate inclusion MIC method of the NNN of Australia. In brief, this method uses an inoculum of 104 cfu spotted on IsoSensitest agar (Oxoid) containing 8% saponin-lysed horse blood and relevant concentrations of antibiotic. CAT activity was assayed spectrophotometrically with a number of modifications from the method of White et al.2 Briefly, a small amount of the test organism grown on chocolate agar was lysed in 100 µL of 10% SDS. Five microlitres of the lysate was added to 50 µL of reagent containing 100 mM Tris HCL (pH 7.5), 1 mM 5-thio-2-nitrobenzoate and 2 mM chloramphenicol; 50 µL 10 mM acetyl coenzyme A was then added. Positive controls were four known CAT-positive isolates of Haemophilus influenzae and coenzyme A, and the negative control was N. meningitidis NCTC 8554. Colour changes were measured spectrophotometrically at 412 nm.

Using PCR, the resistant isolates were tested for the presence of the catP gene from Tn4451, as found in previously isolated CmRNm.1 Oligonucleotide primers specific for the catP gene or for Tn4451 DNA flanking the catP gene were used.9 In this way it could be determined whether catP was present and if the flanking DNA was absent in these isolates, as found previously.1 An additional set of oligonucleotide primers1 was used to investigate the insertion site of Tn4451 DNA in the N. meningitidis chromosome. DNA sequencing was used to confirm the amplified PCR products. A chloramphenicol-susceptible serogroup B N. meningitidis strain MC58 served as the negative control.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Two CmRNm isolates were detected among 1382 strains examined during January 1994–March 2003. Both CmRNm isolates were of serogroup B, although they exhibited different phenotypes (Table 1). Lysates from both organisms were able to acetylate chloramphenicol. Positive and negative CAT assay controls gave appropriate reactions. Disc screening and MIC analysis confirmed that they were chloramphenicol resistant. Different MIC values were obtained by the different methods, but were in the range 16–64 mg/L for the two resistant meningococci (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Characteristics of chloramphenicol-resistant Neisseria meningitidis isolates
 
Using the Tn4451-derived primers specific for catP and the 3' end of the upstream gene tnpV, a product of the expected size was amplified from each of the chloramphenicol-resistant isolates, but not from the chloramphenicol-susceptible negative control strain. This result implied that the catP gene, and at least part of the tnpV gene, was present in the isolates tested. DNA sequencing of the amplified products confirmed the presence of catP and the 3' end of tnpV from Tn4451. Using other oligonucleotide primer pairs specific for DNA flanking catP on Tn4451, products of the expected sizes were not amplified from the CmRNm, suggesting that no other Tn4451 sequences were present in these isolates. This analysis therefore suggests that the isolates contain the same DNA insert as the isolates examined by Galimand et al.1

To confirm the end points of the Tn4451 DNA, and the point of insertion of this DNA into the N. meningitidis chromosome, additional PCRs were performed. Oligonucleotide primers C and D,1 specific for the meningococcal sequences flanking the Tn4451 insertion in the CmRNm isolates examined previously, were used. Since the publication of Galimand et al.,1 sequencing of the genome of the serogroup B N. meningitidis MC58 strain has been completed.10 Analysis of this sequence indicates that the Tn4451 DNA has inserted into a gene designated as TIGR locus NMB1350, encoding a hypothetical protein.

Using oligonucleotide primers C and D,1 larger PCR products of ~1.2 kb were amplified from each of our CmRNm isolates, as compared with the chloramphenicol-susceptible N. meningitidis negative control strain, from which a product of ~0.22 kb was amplified. Sequencing of these products showed that the point of insertion of Tn4451 DNA in the N. meningitidis chromosome was identical in both our chloramphenicol-resistant isolates, and was the same as that seen previously, with no loss of meningococcal DNA upon insertion of the Tn4451 DNA.1


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The two isolates confirmed as CmRNm were phenotypically distinct and separated in time and place. Both were shown to be chloramphenicol resistant, by means of a catP-encoded CAT enzyme. It also seems likely that the same truncated integrative element, described in the 12 previously tested CmRNm,1 is also present in the two Australian isolates. This was not a systematic study of all invasive meningococcal isolates in Australia, so the frequency of this occurrence in Australia is not known precisely. However, the sample of isolates examined (1382) comprised nearly half the samples available to the NNN over a 9 year period, which suggests that the incidence of CmRNm in Australia is low.

The origin of the resistance determinants in these two isolates remains a matter for speculation. The original report by Galimand et al.1 and subsequent analysis gave rise to the view that the resistance determinant, catP, arose in C. perfringens and may have been transferred to N. meningitidis either directly or by one or more intermediate hosts. There was no record in either of these patients of any sustained exposure to antibiotics in general and none to chloramphenicol in particular. Although the patient in case 1 was originally from Vietnam, where some of the reports of this phenomenon were first described, she had been in Australia for some considerable time. In the previous study,1 one of the original 12 patients was from metropolitan France without any known contact with South-East Asia. Antibiotic use in Australia is highly regulated, and systemic chloramphenicol use is extremely uncommon. However, there is a surprisingly high use of topical chloramphenicol in Australia and about 1 380 000 prescriptions for topical ophthalmic preparations for chloramphenicol were filled for a population of about 18 500 000 in Australia in 1997.10 The frequent use of topical ophthalmic preparations of this antibiotic leads to the suggestion that commensal organisms may often be exposed to chloramphenicol by its passage from the eye via the lacrimal apparatus to the oropharynx and intestine. It is also possible that chloramphenicol residues in food, anecdotally reported in seafood and honey, may be associated with this phenomenon. It is established that commensal Neisseria are the likely origin of the mosaic penA genes seen in N. meningitidis less susceptible to penicillin.11 That is, the alteration in penicillin susceptibility now being seen to some extent in meningococci appears to have arisen in other pharyngeal Neisseria and been transferred by genetic exchange to meningococci.12

Serogroup B N. meningitidis are highly transformable, unlike serogroup A N. meningitidis, and it is thus interesting that to date all examples of chloramphenicol resistance through acquisition of a genetically transferable element have been in this serogroup and into precisely the same chromosomal location. Analysis of the serogroup A19 genome sequence clearly indicates the presence of this chromosomal sequence. Sequencing the meningococcal C genome (Wellcome Trust Sanger Institute, Cambridge, UK) is nearing completion and it would be interesting to determine if this genome also contains the sequence. If it is indeed the case that this type of resistance is more likely to transfer to serogroup B organisms, it provides an explanation for the lack of resistance to chloramphenicol noted in areas where the non-transformable serogroup A meningococci are common.

However, there is no established method for screening for resistance to these agents. Some test systems do not recommend the use of disc screening methods for meningococci,13 whereas others have established reliable and valid systems using these easily applied and cheap methods.8,14 The limited experience with resistant organisms derived here with one of these methods suggests that disc testing may indeed be suitable for screening for strains of this type. It is also noted that there was some discrepancy between MIC values obtained with different test methods in these strains and between the results obtained here and in France. The MICs for the two Australian isolates were in the range 16–64 mg/L by various standard methods, whereas those tested in France were in the range 64–192 mg/L. It is not known whether the different test methods themselves account for this range or if the variation is due to different levels of enzyme production. This may be important because the clinical relevance of this phenomenon has yet to be established. This will require a correlation between clinical outcome and MIC that is difficult to establish in diseases such as invasive meningococcal syndromes. Despite this lack of first-hand evidence, it would seem prudent to assume that appearance of organisms with this form of resistance in areas with a high meningococcal disease burden would require the alteration of programmatic treatments based on chloramphenicol preparations.


    Acknowledgements
 
Dr Mark Achtman (Department of Molecular Biology, Max-Planck Institut für Infektionsbiologie, Berlin, Germany) kindly supplied the control strain MC58 and this and other isolates were phenotyped by staff at the South Western Area Pathology Services. The National Neisseria Network was supported financially by the Department of Health and Ageing of the Commonwealth Government of Australia. Work at Monash University was supported by grants from the Australian National Health and Medical Research Council.


    Footnotes
 
* Corresponding author. Tel: +612-9382-9079; Fax: +612-9389-9098; E-mail: j.tapsall{at}unsw.edu.au Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1 . Galimand, M., Gerbaud, G., Guibourdenche, M. et al. (1998). High-level chloramphenicol resistance in N. meningitidis. New England Journal of Medicine 339, 868–74.[Abstract/Free Full Text]

2 . White, P. A., Stokes, H. W., Bunny, K. L. et al. (1999). Characterisation of a chloramphenicol acetyltransferase determinant in the chromosome of Pseudomonas aeruginosa. FEMS Microbiology Letters 175, 27–35.[CrossRef][ISI][Medline]

3 . Adams, V., Lyras, D., Farrow, K. et al. (2002). The clostridial mobilisable transposons. Cell and Molecular Life Sciences 59, 2033–43.[ISI]

4 . Nicolas, P., Raphenon, G., Guibourdenche, M. et al. (2000). The 1998 Senegal epidemic of meningitis was due to the clonal expansion of A:4:P1.9, clone III-1, sequence type 5 Neisseria meningitidis strains. Journal of Clinical Microbiology 38, 198–200.[Abstract/Free Full Text]

5 . Blondeau, J. & Yaschuk Y. (1995). In vitro activities of ciprofloxacin, cefotaxime, ceftriaxone, chloramphenicol, and rifampicin against fully susceptible and moderately penicillin-resistant Neisseria meningitidis. Antimicrobial Agents and Chemotherapy 39, 2577–9.[Abstract]

6 . Pascual, A., Joyanes, P., Martinez-Martinez, L. et al. (1996). Comparison of broth microdilution and E-test for susceptibility testing of Neisseria meningitidis. Journal of Clinical Microbiology 34, 588–91.[Abstract]

7 . Tondella, M.-L., Rosenstein, N., Mayer, L. et al. (2001). Lack of evidence for cloramphenicol resistance in Neisseria meningitidis, Africa. Emerging Infectious Diseases 7, 163–4.[ISI][Medline]

8 . Bell, S. M., Gatus, B. J., Pham, J. N. et al. (2002). Antibiotic Susceptibility Testing by the CDS Method. A Manual for Medical and Veterinary Laboratories. Australian Society for Microbiology, Melbourne, Australia.

9 . Lyras, D. & Rood J. I. (2000). Transposition of Tn4451 and Tn4453 involves a circular intermediate that forms a promoter for the large resolvase, TnpX. Molecular Microbiology 38, 588–601.[CrossRef][ISI][Medline]

10 . Tettelin, H., Saunders, N. J., Heidelberg, J. et al. (2000). Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287, 1809–15.[Abstract/Free Full Text]

11 . Commonwealth Department of Health and Family Services. (1998). Australian Statistics on Medicines, p. 157. Commonwealth of Australia, Canberra, Australia.

12 . Bowler, L. D., Zhang, Q. Y., Riou, J. Y. et al. (1994). Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory stimulation. Journal of Bacteriology 176, 333–7.[Abstract]

13 . National Committee for Clinical Laboratory Standards. (2002). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A5. NCCLS, Villanova, PA, USA.

14 . British Society for Antimicrobial Chemotherapy. (2003). BSAC disc diffusion method for antimicrobial susceptibility testing. Version 2.1.3. [Online.] http://www.bsac.org.uk/ (15 May 2003, date last accessed).