Role of penA polymorphisms for penicillin susceptibility in Neisseria lactamica and Neisseria meningitidis

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Abstract

In meningococci, reduced penicillin susceptibility is associated with five specific mutations in the transpeptidase region of penicillin binding protein 2 (PBP2). We showed that the same set of mutations was present in 64 of 123 Neisseria lactamica strains obtained from a carriage study (MIC range: 0.125–2.0 mg/L). The PBP2 encoding penA alleles in these strains were genetically similar to those found in intermediate resistant meningococci suggesting frequent interspecies genetic exchange. Fifty-six N. lactamica isolates with mostly lower penicillin MICs (range: 0.064–0.38 mg/L) exhibited only three of the five mutations. The corresponding penA alleles were unique to N. lactamica and formed a distinct genetic clade. PenA alleles with no mutations on the other hand were unique to meningococci. Under penicillin selective pressure, genetic transformation of N. lactamica penA alleles in meningococci was only possible for alleles encoding five mutations, but not for those encoding three mutations; the transfer resulted in MICs comparable to those of meningococci harboring penA alleles that encoded PBP2 with five mutations, but considerably lower than those of the corresponding N. lactamica donor strains. Due to a transformation barrier the complete N. lactamica penA could not be transformed into N. meningitidis. In summary, penicillin MICs in N. lactamica were associated with the number of mutations in the transpeptidase region of PBP2. Evidence for interspecific genetic transfer was only observed for penA alleles associated with higher MICs, suggesting that alleles encoding only three mutations in the transpeptidase region are biologically not effective in N. meningitidis. Factors other than PBP2 seem to be responsible for the high levels of penicillin resistance in N. lactamica. A reduction of penicillin susceptibility in N. meningitidis by horizontal gene transfer from N. lactamica is unlikely to happen.

Introduction

Neisseria meningitidis (N. meningitidis, meningococci) is a facultative pathogenic bacterium that asymptomatically colonizes the nasopharynx of approximately 10% of the human population (Gold et al., 1978, Cartwright et al., 1987). Occasionally, N. meningitidis causes severe diseases like meningitis and septicemia (Rosenstein et al., 2001). The apathogenic species N. lactamica is closely related to meningococci and shares the same habitat (Guibourdenche et al., 1986). However, N. lactamica colonizes predominately infants and toddlers, whereas carriage of meningococci is much more frequent in adolescents and young adults (Gold et al., 1978, Cartwright et al., 1987, Bennett et al., 2005, Kristiansen et al., 2012).

Penicillin G remains a first-line agent against invasive meningococcal infections (Nadel and Kroll, 2007), but in the last years the number of meningococcal isolates that displayed reduced penicillin susceptibility increased in Europe and other parts of the world (Richter et al., 2001, Tapsall et al., 2001, Vazquez et al., 2007, Bertrand et al., 2012, Bijlsma et al., 2014). Between 2002 and 2007, 14.1% of the invasive meningococci in Germany were intermediate susceptible or resistant towards penicillin (Vogel and Frosch, 2008). Five specific amino acid polymorphisms in the transpeptidase region of penicillin binding protein 2 (PBP2), encoded by the penA gene, showed high correlation with reduced penicillin susceptibility (Thulin et al., 2006, Taha et al., 2007). Commensal Neisseria spp. have been suggested to be a source for altered penA alleles (Spratt et al., 1989, Spratt et al., 1992, Saez-Nieto et al., 1990, Lujan et al., 1991). In a Spanish study, most N. lactamica isolates showed decreased susceptibility to penicillin (Arreaza et al., 2002). There are, however, no data on penA sequences of N. lactamica.

In this study, penicillin susceptibility and penA alleles were analyzed for 123 N. lactamica and 129 meningococcal strains isolated in Germany. Furthermore, transformation experiments were conducted to investigate the effect of penA transfer from N. lactamica to N. meningitidis.

Section snippets

Bacterial isolates and antimicrobial resistance testing

N. lactamica strains (n = 123) were collected during the Bavarian meningococcal carriage study in winter 1999/2000 (Alber et al., 2001). N. meningitidis strains (n = 129) were randomly selected from invasive isolates received by the German reference laboratory for meningococci in 2006. For transformation experiments the capsule null locus (cnl) meningococcal strain α14 (Claus et al., 2002, Schoen et al., 2008) was used.

MICs of penicillin G were determined with Etest® strips (bioMérieux, Nürtingen,

Penicillin susceptibility of N. lactamica and N. meningitidis

In total, 123 N. lactamica strains and 129 invasive meningococci were analyzed by Etest® to determine their penicillin G susceptibility. MIC values in N. lactamica (range: 0.064–2.0 mg/L, MIC50 = 0.38) were significantly higher than those in N. meningitidis (range: 0.016–0.25 mg/L, MIC50 = 0.064) (p  0.001) (Fig. 1A, Table 2). Forty-five of the N. lactamica strains, for which β-lactamase production was ruled out, but none of the meningococcal strains were resistant to penicillin. Only three N.

Discussion

For a collection of Spanish N. lactamica isolates it was shown that penicillin MIC values were higher than those found in meningococci (Arreaza et al., 2002). These results could be confirmed in the present study for a German strain collection, suggesting that at least in Europe penicillin MICs are higher in N. lactamica than in N. meningitidis. Genetic analysis of the 3′ region of the penA gene revealed that it was much more variable in N. lactamica than in meningococci and both species had

Acknowledgements

The German reference laboratory for meningococci and Haemophilus influenzae is supported by the Robert Koch-Institute with funds of the German Federal Ministry of Health (funding code 1369-237). Moreover, we are grateful to Eva Hong, the curator of the penA database. This publication made use of the Neisseria Multi Locus Sequence Typing website (http://pubmlst.org/ neisseria/) developed by Keith Jolley and sited at the University of Oxford (Jolley and Maiden, 2010). The development of this site

References (47)

  • S. Bertrand et al.

    Evolutionary changes in antimicrobial resistance of invasive Neisseria meningitidis isolates in Belgium from 2000 to 2010: increasing prevalence of penicillin nonsusceptibility

    Antimicrob. Agents Chemother.

    (2012)
  • K.A. Cartwright et al.

    The Stonehouse survey: nasopharyngeal carriage of meningococci and Neisseria lactamica

    Epidemiol. Infect.

    (1987)
  • H. Claus et al.

    Many carried meningococci lack the genes required for capsule synthesis and transport

    Microbiology

    (2002)
  • H. Claus et al.

    Genetic analysis of meningococci carried by children and young adults

    J. Infect. Dis.

    (2005)
  • Clinical and Laboratory Standards Institute

    Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement. M100-S24

    (2014)
  • G. Di Lallo et al.

    Use of a two-hybrid assay to study the assembly of a complex multicomponent protein machinery: bacterial septosome differentiation

    Microbiology

    (2003)
  • C. Fraipont et al.

    The integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli

    Microbiology

    (2011)
  • R. Gold et al.

    Carriage of Neisseria meningitidis and Neisseria lactamica in infants and children

    J. Infect. Dis.

    (1978)
  • K.E. Hagman et al.

    Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system

    Microbiology

    (1995)
  • D.H. Huson

    SplitsTree: analyzing and visualizing evolutionary data

    Bioinformatics

    (1998)
  • D.H. Huson et al.

    Application of phylogenetic networks in evolutionary studies

    Mol. Biol. Evol.

    (2006)
  • K.A. Jolley et al.

    BIGSdb: Scalable analysis of bacterial genome variation at the population level

    BMC Bioinf.

    (2010)
  • G. Karimova et al.

    Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis

    J. Bacteriol.

    (2005)
  • Cited by (0)

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