ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2004, p. 3103–3106
0066-4804/04/$08.00?0 DOI: 10.1128/AAC.48.8.3103–3106.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 8
Emergence in Italy of a Neisseria meningitidis Clone with Decreased
Susceptibility to Penicillin
Paola Stefanelli, Cecilia Fazio, Arianna Neri, Tonino Sofia, and Paola Mastrantonio*
Department of Infectious, Parasitic and Immuno-Mediated Diseases, Istituto Superiore di Sanita’, Rome, Italy
Received 8 March 2004/Returned for modification 6 April 2004/Accepted 13 April 2004
A rise in invasive diseases due to Neisseria meningitidis C:2b:P1.5 with decreased penicillin susceptibility
occurred in Italy during the last 2 years. Real-time PCR identified the Peniphenotype, and the penA sequence
revealed the mosaicism of the gene. Molecular analyses assigned the isolates to a single emergent clone of the
hypervirulent A4 cluster.
In Italy, the incidence of meningococcal disease has been
consistently low (about 0.3 to 0.4 per 100,000 inhabitants per
year). Throughout the 1990s, Neisseria meningitidis serogroup
C strains represented fewer than 30% of all isolated meningo-
cocci. In 2002 to 2003, we observed an increase in serogroup C
meningococcal disease, which became responsible for 42.5% of
culture-confirmed cases. Interestingly, most of these isolates
have the antigenic phenotype C:2b:P1.5 and show intermediate
susceptibility to penicillin (Peni) (0.06 ?g/ml ? MIC ? 1 ?g/
ml), due to sequence changes in the penicillin binding protein
2 penA gene. Penimeningococci have been reported and are
being monitored in several countries (3, 5, 8). The attention is
driven by the concept that strains with decreased susceptibility
may still be evolving and in the process of acquiring further
alterations in the penA gene, anticipating the appearance of
resistant strains, as observed for Streptococcus pneumoniae and
Neisseria gonorrhoeae (9, 12). In Italy, before 2002 Penimenin-
gococci accounted for 7.5% of the isolates, but since then the
percentage has risen significantly (to 27.4%). All Penistrains
showed a mosaic structure in the transpeptidase region of the
penA gene (1), and a recently validated real-time PCR assay
(10) has been used for rapid confirmation of this phenotype. In
this study, all C:2b:P1.5 Penimeningococci isolated in Italy
were analyzed by penA sequencing, multilocus sequence typ-
ing, and pulsed-field gel electrophoresis (PFGE) to determine
whether the recent increase in the number of cases due to this
phenotype correlates with the emergence of a single clone and
whether it circulates in other European countries.
A total of 214 invasive N. meningitidis strains were received
at the reference laboratory of the National Surveillance of
Meningococcal Meningitis between January 2002 and Decem-
ber 2003. All isolates were typed (7), and of 214 N. meningitidis
isolates received, 91 were serogroup C and 38 of these (42.2%)
belonged to phenotype C:2b:P1.5. Among the remaining sero-
group C strains, 21% had the phenotype C:2a:P1.5, which up to
then was the most frequent phenotype among C strains, and
the others showed a variety of different sero- and subtypes.
The recently described real-time PCR (10) was used to
discriminate between penicillin-susceptible (Pens) and Peni
strains. Two hybridization probes were used to distinguish the
wild-type penA gene in the Pensmeningococci from the mu-
tated gene at codon 566 in Penistrains. Thermal analysis of
probe hybridization revealed melting temperatures of 45.5 and
55°C for the Pensand Penistrains, respectively, as shown in
Fig. 1A. MICs of penicillin were also assessed by use of the
E-test (AB Biodisk) on Mueller-Hinton agar (Oxoid) supple-
mented with 5% sheep blood, and the majority of C:2b:P1.5
strains (78.4%) showed MICs of ?0.094 ?g/ml, whereas a few
serogroup C strains (24%) with other sero- or subtypes were
The Peniphenotype was finally confirmed by sequencing the
penA PCR products in the transpeptidase domain of the en-
coding gene. Analysis of the sequences was performed with the
Accelrys Wisconsin Genetics Computer Group package.
The penA sequence analysis showed a short DNA region,
between nucleotides 1364 and 1545, with 98% identity to the
sequence derived from Neisseria perflava/sicca (accession num-
ber X76422) and 100% homology with all penA sequences
from Spanish C:2b:P1.5,2 Penistrains deposited in the Na-
tional Center for Biotechnology Information databank (http:
//www.ncbi.nlm.nih.gov). Conversely, the penA genes of Peni
meningococci with other serotypes or serosubtypes appear to
have acquired larger blocks of DNA, often from more than one
commensal Neisseria species along all of the transpeptidase
domain (Fig. 1B). The replacement of the short region be-
tween nucleotides 1364 and 1545 in the C:2b:P1.5 strains seems
to be sufficient to produce a form of the protein with a lower
affinity to penicillin.
The spread of PeniC:2b:P1.5 meningococci over the country
was more remarkable in the first 6 months of 2003, when twice
as many were isolated as in the previous year. This sharp
increase led us to carry out a molecular analysis of these strains
to confirm the circulation of a clone. According to multilocus
sequence typing results, obtained by the methodology de-
scribed by Maiden et al. (6) (http://neisseria.org/nm/typing
* Corresponding author. Mailing address: Department of Infectious,
Parasitic & Immuno-mediated Diseases, Istituto Superiore di Sanita’,
Viale Regina Elena 299, 00161-Rome, Italy. Phone: 390649902335.
Fax: 390649387112. E-mail: firstname.lastname@example.org.
FIG. 1. (A) Examples of Tmcurves for Pensand PeniN. meningitidis phenotypes obtained by real-time PCR assay with the mutated 566 codon
in the penA gene. (B) Mosaic penA genes of PeniN. meningitidis strains. Each line indicates the proposed origin of the exogenous DNA blocks
in the transpeptidase domain of the gene. The arrowheads show the position of the mutated 566 codon used as a marker of penA translocation
in the real-time PCR assay. nt, nucleotides; AC#, accession number.
3104 NOTESANTIMICROB. AGENTS CHEMOTHER.
/mlst/), all of the C:2b:P1.5 Penimeningococci were assigned to
the ST8/A4 cluster (Table 1), which is one of the two hyper-
virulent lineages responsible for most of serogroup C disease
worldwide (the other is ST11). DNA macrorestriction frag-
ments generated with the NheI restriction enzyme and ana-
lyzed by PFGE, as previously described (4), showed one main
pulse type (PTA) and two subclones with two or three minor
differences attributable to point mutations, named PTA1 and
PTA2 (Fig. 2). When BglII was used, all of the strains showed
the same pulse type (data not shown). One Penistrain with
phenotype C:2b:nst, isolated in the first months of 2003, be-
longed to the same clone. The lack of identification of a sero-
subtype in this strain was due to the presence of IS1301 in the
porA gene and its subsequent inactivation, as demonstrated by
sequencing (data not shown).
Interestingly, the pulse type PTA found in most of the ex-
amined C:2b:P1.5 strains is identical to fingerprint pattern 2
(PT2) described by Arreaza et al. (2) for C:2b:P1.5,2 epidemic
Spanish strains, just as our PTA1 resembles PT1 described in
the same paper. Unlike the Spanish PT1 strains, the PTA1
strains are able to cause disease in all age groups and not
predominantly in children under 2 years of age. In contrast, the
subclone PTA2 is a unique profile found only in two strains,
both of which were responsible for meningitis in infants.
All of these findings seem to confirm that the recent increase
of meningococcal disease caused by N. meningitidis C:2b:P1.5
in Italy is due to the spread of a single emergent clone with
decreased penicillin susceptibility and belonging to the hyper-
virulent cluster A4. Although the first strain C:2b:P1.5, ST8/A4
cluster appeared in Italy in 1998, in the following 3 years all
meningococci with phenotype C:2b:P1.5 belonged to ST1860, a
new ST of the ET37 complex detected in Italy (11). None of
these strains were Peni, in contrast to most of the serogroup C
ET37 strains isolated in other countries. In 2002, Penimenin-
gococci of phenotype C:2b:P1.5, ST8/A4 cluster suddenly re-
appeared and rapidly spread all over the country.
We speculate that the Spanish clone might have been im-
ported in the second half of the 1990s. In fact, one Penistrain
with phenotype C:2b:P1.5,2 ST8/A4 and pulse type PTA1 was
isolated in Italy in 1996, and it showed the same molecular
characteristics as the Spanish strains. After this episode, for
reasons difficult to understand and linked to the evolution and
dynamics of the meningococcal population, it took some years
to settle in, with a minor modification in the outer membrane
proteins encoded by the porA gene. However, once established,
the phenotype C:2b:P1.5 has become so fit as to spread very
rapidly and to successfully compete with the other phenotypes.
The acquisition of a very small exogenous DNA fragment
might have provided an advantage in terms of fitness compared
to the other Peniphenotypes. It is important to underline that
there is a direct relationship between the increase in serogroup
C strains causing meningococcal disease in Italy and the eight-
fold increase of meningococci with decreased susceptibility to
penicillin as a result of the spread of this virulent clone. It will
be of extreme importance to closely monitor the endemic cir-
culation of this phenotype in order to plan specific vaccination
programs in a timely fashion.
We thank the microbiologists of the hospital laboratories participat-
ing in the Italian National Surveillance of Bacterial Meningitis for
isolating the strains and sending them to the Reference Laboratory of
the Istituto Superiore di Sanita’ (Rome). This study made use of the
Neisseria MultiLocus Sequence Typing website (http://neisseria.mlst
.net) developed by Man-Suen Chan and sited at the University of
The development of the Neisseria MultiLocus Sequence Typing
website is funded by the Wellcome Trust. This work was partially
funded by Ministero della Salute (Italy), Programma per la Ricerca
FIG. 2. PFGE profiles of genomic DNAs from N. meningitidis
C:2b:P1.5 strains after digestion with endonuclease NheI. The lambda
ladder DNA marker (New England Biolabs) (lane MW) was used as a
molecular size standard (48.5 kb). Lane 1, pulse type PTA; lane 2,
PTA1; lane 3, PTA2. The arrows identify the band differences.
TABLE 1. Characteristics of N. meningitidis C:2b:P1.5 strains
belonging to the ST8/A4 cluster isolated between
January 2002 and December 2003
3, 4, 17, 19
?1, 4, 15, 24, 49, NKa
4, 11, 12, 17, 35
8, 30, 60, 62
aNK, not known.
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