JAC
Journal of Antimicrobial Chemotherapy (2002) 49, 545–547
What about antibiotic resistance in Neisseria lactamica? L. Arreaza, C. Salcedo, B. Alcalá and J. A. Vázquez* Servicio de Bacteriología, Laboratorio de Referencia de Meningococos, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain The in vitro activity of penicillin, ampicillin, cefotaxime, ceftriaxone, rifampicin and ciprofloxacin against 286 Neisseria lactamica isolates was determined by agar dilution and the category of susceptibility was analysed in accordance with the criteria used for Neisseria meningitidis. All isolates were considered to have intermediate susceptibility to penicillin. A total of 1.7% of the isolates were resistant to ampicillin but all were susceptible to cefotaxime and ceftriaxone. Rifampicin MICs ranged between 0.12 and 2 mg/L. Six isolates (2.1%) showed decreased susceptibility to ciprofloxacin.
Introduction Neisseria lactamica is part of the commensal bacterial flora of the human upper respiratory tract and shares this ecological niche with Neisseria meningitidis. Like other Neisseria species, N. lactamica is naturally transformable and can transfer antibiotic resistance markers into the closely related species N. meningitidis.1 There are few studies analysing the susceptibility of this species to the antimicrobial drugs frequently used in the treatment and prophylaxis of meningococcal disease (MD).2 The role of N. lactamica in the emergence of N. meningitidis strains with intermediate resistance to penicillin (Peni), by horizontal genetic exchange of penicillin binding protein (PBP) genes, has been proposed in several studies.2,3 Intragenic recombination of the meningococci chromosomal gene penA, encoding the target site for penicillin, with related genes of N. lactamica and other commensal Neisseria spp. may have generated mosaic genes that encode proteins with reduced affinity for penicillin, resulting in the emergence of Peni meningococcal strains.4 The aim of this study was to analyse the levels of susceptibility of N. lactamica isolates to antimicrobial agents commonly used for treatment and prophylaxis of MD.
between 1996 and 1998, were studied.5 All isolates were identified as N. lactamica by standard methods.2 Susceptibility to penicillin, ampicillin, cefotaxime, ceftriaxone, rifampicin and ciprofloxacin was determined by the agar dilution method in Mueller–Hinton agar (Difco Laboratories, Detroit, MI, USA) supplemented with 5% sheep blood, with a final inoculum of 104–105 cfu per spot. The doubling dilution ranges tested were 0.007–16 mg/L for penicillin and ampicillin, 0.0003–1 mg/L for cefotaxime and ceftriaxone, 0.007–2 mg/L for rifampicin and 0.0007–1 mg/L for ciprofloxacin. Plates were incubated for 20 h at 37C in air containing 5% CO2. The MIC was defined as the lowest concentration at which no growth was visible. The categories of ‘susceptible’, ‘intermediate’ and ‘resistant’ were defined according to the criteria used for N. meningitidis.4 Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used as quality control organisms and were included each time isolates were tested. In order to investigate the possible role of membrane permeability in the susceptibility of N. lactamica to rifampicin the MICs of this drug were determined in the presence and absence of Tween 80 (0.05%) for 41 isolates according to the protocol described previously.6
Results and discussion
Material and methods Two hundred and eighty-six N. lactamica isolates obtained from two meningococcal carrier surveys undertaken by the Dirección Xeral de Saúde Pública (Xunta de Galicia) and the Spanish Reference Laboratory for Meningococci,
The Table shows the susceptibility of the isolates to the antimicrobial drugs tested as well as the criteria used to define the categories of susceptibility or resistance. All the isolates appeared intermediately resistant to penicillin (Peni), with MICs between 0.12 and 1 mg/L. Most of the
*Corresponding author. Tel: 34-91-5097901; Fax: 34-91-5097966; E-mail:
[email protected]
545 © 2002 The British Society for Antimicrobial Chemotherapy
L. Arreaza et al. Table. Antimicrobial susceptibility of N. lactamica isolates (n 286) MIC (mg/L)
No. (%) of isolates
Antibiotic
range
MIC50
MIC90
susceptible
intermediate
resistant
Penicillina Ampicillina Cefotaximeb Ceftriaxonec Rifampicind Ciprofloxacine
0.12–1 0.25–2 0.0015–0.25 0.0007–0.06 0.12–2 0.0015–0.5
0.25 0.5 0.015 0.0015 0.5 0.003
0.5 1.0 0.06 0.015 1.0 0.007
0 0 286 (100) 286 (100) 284 (99.3) 280 (97.9)
286 (100) 281 (98.3) –f – 2 (0.7) 6 (2.1)
0 5 (1.7) – – 0 0
MICs susceptible 0.06 mg/L; intermediate 0.12–1 mg/L; resistant 2 mg/L. MICs susceptible 0.5 mg/L. c MICs susceptible 0.25 mg/L. d MICs susceptible 1 mg/L; intermediate 2 mg/L; resistant 4 mg/L. e MICs susceptible 0.06 mg/L; intermediate 0.12–0.5 mg/L; resistant 1 mg/L. f Category not determined. a
b
isolates had ampicillin MICs two-fold higher than those of penicillin. This phenomenon is frequent in N. meningitidis7 and five strains showed resistance to this antibiotic (MIC 2 mg/L) according to criteria used previously.7,8 All the isolates were susceptible to cefotaxime and ceftriaxone, although the MICs were higher than those found for meningococci.7 The high proportion of Peni N. lactamica strains could explain this finding. Slight increases in the MICs of the broad-spectrum cephalosporins, such as cefotaxime and ceftriaxone, for Peni N. meningitidis strains have been described previously.9 The finding that all the isolates of N. lactamica were Peni shows that this level of resistance appeared in this species before it emerged in N. meningitidis. The mosaic genes described for meningococcal strains have arisen as a result of horizontal genetic exchange involving penA genes particularly from Neisseria flavescens, but also from Neisseria cinerea, Neisseria mucosa and N. lactamica, which are closely related species.1,2,4 Penicillin resistance in meningococci may be the result of different and separate recombination events, as many different mosaic penA alleles have been found in Peni meningococci.1 A comparison of the penA sequences in N. lactamica isolates with the sequences in other Neisseria species would clearly merit investigation. Most of the isolates were susceptible to rifampicin. MIC50 and MIC90 of rifampicin were higher than those described for N. meningitidis,7 although no isolate was resistant. Changes in the susceptibility to rifampicin may be because of alterations in membrane permeability,6 so differences in the membrane composition between N. meningitidis and N. lactamica might be the explanation for the differences in the susceptibility levels to rifampicin. Tween 80 is a detergent that has been used to investigate membrane permeability in N. meningitidis.6 In the study of Abadi et al.,6 no difference was observed with meningo-
coccal strains with intermediate levels of resistance. However, we nonetheless decided to use Tween 80 for N. lactamica isolates with intermediate resistance to rifampicin because N. meningitidis might have a different membrane composition in comparison with N. lactamica. No difference in growth rates in the presence and absence of Tween 80 was observed for isolates with different MICs. Point mutations in the rpoB gene in N. lactamica could also account for the higher rifampicin MICs seen for N. lactamica compared with meningococci.7 Sequencing of this gene in N. lactamica and its comparison with the rpoB sequence in N. meningitidis (susceptible and resistant isolates) should be undertaken to resolve this issue. Finally, six isolates showed intermediate susceptibility to ciprofloxacin, with MICs of 0.12 mg/L (n 4) and 0.5 mg/L (n 2). Although isolates with this level of susceptibility are frequent in N. gonorrhoeae,4 such isolates have not been described among those Neisseria species that share an ecological niche with N. meningitidis. Such N. lactamica strains might constitute a potential DNA source for the development of ciprofloxacin resistance in N. meningitidis. To date only two meningococcal strains with decreased susceptibility to ciprofloxacin have been described.10 Further studies will be necessary in order to determine whether these N. lactamica strains show changes in gyrA and parC genes similar to those described in gonococci. Transformation experiments with the DNA of N. lactamica strains with decreased susceptibility to ciprofloxacin and susceptible meningococci isolates could demonstrate whether horizontal spread of ciprofloxacin resistance between meningococci and N. lactamica can easily happen. Horizontal genetic exchange of antimicrobial resistance determinants between commensal Neisseria species, such as N. lactamica and N. meningitidis is, perhaps, one of the most important mechanisms in the continuing evolution of resistance to antimicrobial agents in N. meningitidis.4
546
Antibiotic resistance in Neisseria lactamica
Acknowledgements C.S. and B.A. were supported by predoctoral fellowships from ISCIII and Fondo de Investigación Sanitaria (FIS) 98/0311. L.A. was supported by a postdoctoral fellowship from Instituto de Salud Carlos III.
References 1. Maiden, M. C. J. (1998). Horizontal genetic exchange, evolution, and spread of antibiotic resistance in bacteria. Clinical Infectious Diseases 27, Suppl. 1, 12–20. 2. Sáez-Nieto, J. A., Luján, R., Matinez-Suarez, J. V., Berrón, S., Vázquez, J. A., Viñas, M. et al. (1990). Neisseria lactamica and Neisseria polysaccharea as possible sources of meningococcal β-lactam resistance by genetic transformation. Antimicrobial Agents and Chemotherapy 34, 2269–72. 3. Luján, R., Zhang, Q. Y., Sáez-Nieto, J. A., Jones, D. M. & Spratt, B. G. (1991). Penicillin resistant isolates of Neisseria lactamica produced altered forms of penicillin binding protein 2 that arose by interspecies gene transfer. Antimicrobial Agents and Chemotherapy 35, 300–4. 4. Vázquez, J. A. (2001). The resistance of Neisseria meningitidis to the antimicrobial agents: an issue still in evolution. Reviews in Medical Microbiology 12, 39–45.
5. Arreaza, L., Berrón, S., Fernández, S., Santiago, M. I., Malvar, A. & Vázquez, J. A. (2000). Investigation for a more virulent variant among the C:2b:P1.2,5 Spanish meningococcal epidemic strains by molecular epidemiology. Journal of Medical Microbiology 49, 1079–84. 6. Abadi, F. J. R., Carter, P. E., Cash, P. & Pennington, T. H. (1996). Rifampin resistance in Neisseria meningitidis due to alterations in membrane permeability. Antimicrobial Agents and Chemotherapy 40, 646–51. 7. Arreaza, L., de la Fuente, L. & Vázquez, J. A. (2000). Antibiotic susceptibility patterns of Neisseria meningitidis isolates from patients and asymptomatic carriers. Antimicrobial Agents and Chemotherapy 44, 1705–7. 8. Baquero, F., Martínez Beltrán, J. & Cantón, R. (1997). MENSURA break-points. Spanish criteria for determination of susceptibility to antibiotics. Revista Española de Quimioterapia 10, 303–13. 9. Sáez-Nieto, J. A., Luján, R., Berrón, S., Campos, J., Viñas, M., Fusté, C. et al. (1992). Epidemiology and molecular basis of penicillin-resistant Neisseria meningitidis in Spain: A 5-year history (1985–1989). Clinical Infectious Diseases 14, 394–402. 10. Shultz, T. R., Tapsall, J. W., White, P. A. & Newton, P. J. (2000). An invasive isolate of Neisseria meningitidis showing decreased susceptibility to quinolones. Antimicrobial Agents and Chemotherapy 44, 1116. Received 11 July 2001; returned 19 September 2001; revised 7 November 2001; accepted 29 November 2001
547
