Transcript vs serogroup Y strains belonging to ST

Prevalence of ST-23 complex among serogroup Y
meningococci isolated from recent cases
of meningitis and septicaemia in Italy
Paola Stefanelli, Cecilia Fazio, Arianna Neri, Tonino Sofia, Paola Mastrantonio
Dept. of Infectious, Parasitic & Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy
Phone: 0039 06 49902126 - email: [email protected]
MATERIALS AND METHODS
INTRODUCTION
In
Italy
two
meningococcal
serogroups are commonly associated
with the disease, namely B and C,
representing
68.1%
and
25.5%
respectively of all isolates during
2007. Cases due to N. meningitidis
strains belonging to serogroup Y are,
on the contrary, very rare. However,
meningitis and septicaemia cases due
to this serogroup have increased in
the country, particularly in 2006.
Interestingly, over the last decade
different countries have reported an
increase in the incidence of invasive
meningococcal disease caused by N.
meningitidis serogroup Y. In order to
gain a better understanding of
serogroup Y meningococcal disease in
Italy, we characterized those isolates
over a three-years period from 2004
to 2006.
Bacterial strains and serotyping
During the period of 2004-2006, 454 N.
meningitidis isolates were received by
the Reference Laboratory (RL) at the
Istituto Superiore di Sanità. Patient
data, including age, sex, provenance was
obtained from specific forms. The strains
were serogrouped by slide agglutination
with commercial antisera (Oxoid). The
serogroup was confirmed by a multiplex
PCR of the siaD gene. Serotypes and
serosubtypes
were
determined
by
standard
whole-cell
ELISA
using
monoclonal antibodies (purchased from
NIBSC, UK) following the standard
method.
PFGE
The
bacterial
DNAs
were
digested with 30 U of NheI (
NewEnglad ,Biolabs) restriction
endonuclease overnight at 37°C.
A 24h electrophoresis was done
in
the
CHEF-MAPPER
II
apparatus (Bio-Rad) with the
following parameters: voltage of
4.5V/cm, pulse time of 1s to
30s,time of run 24 h. The gel was
stained
with
0.5
mg/L
of
ethidium bromide, exposed to
ultraviolet
light,
and
photographed. Tenover’s criteria
(Ref.) were followed to classify
the strains.
MLST
Multilocus Sequence Typing of seven
genes (abcZ, adk, aroE, fumC, gdh,
pdhC,
pgm)
was
performed
as
described by Maiden et al. Primers,
determination of sequence alleles and
designation of sequence types are
described
on
the
MLST
website
(http://neisseria.org/typing/mlst).
Susceptibility to penicillin G
Susceptibility to penicillin G was
determined in Mueller-Hinton agar
supplemented with 5% of sheep
blood using the E-test method. The
intermediate breakpoint for penicillin
was >0.064 to < 1 mg/L
RESULTS
Thirty-five percent of serogroup Y isolates showed a decreased susceptibility to
penicillin (MIC 0.094 mg/L). All of them belonged to the ST23/Cluster A3 (Figure 2)
4
Serogroup Y strains
different from ST23/A3
3
N° of strains
In Italy, the annual average of Neisseria meningitidis serogroup Y strains
has increased during the last years. In 2006 it accounted for 7.1% of all
isolates.
Patients with serogroup Y meningococci were older than patients infected
by other serogroups, with a median age of 34 years. Nearly 50% of the
patients had septicaemia. Over 73% of the isolates were Y:14:NST or
Y:4,14:NST (Table 1)
Serogroup Y strains
ST23/A3
2
Phenotype
Month-Year of
Age (y)
isolation
Clinical presentation
st
Clonal Conplex
Figure 2. MIC values for
penicillin G obtained for
serogroup Y meningococci
isolated in Italy from 2004
to 2006
1
Y:14:NST
2-’04
n.a.1
n.a.
23
ST23/ClusterA3
Y:15:P1.5
3-’04
12
Septicaemia
168
ST-167 complex
Y:14:NST
3-’04
3
Other
23
ST23/ClusterA3
Y:14:NST
3-’04
79
Septicaemia
23
ST23/ClusterA3
Y:14:NST
4-’04
18
Meningitis
23
ST23/ClusterA3
Y:15:P1.16
10-’04
74
Meningitis
1466
ST-174 complex
Y:14:NST
4-’05
52
Meningitis
3974
ST23/ClusterA3
Y:14:NST
5-’05
8
Meningitis
3171
ST23/ClusterA3
Y:4,14:NST
2-’06
70
Meningitis
3171
ST23/ClusterA3
Y:4,14:NST
3-’06
3
Septicaemia
3171
ST23/ClusterA3
Y:14:NST
5-’6
59
Septicaemia
3171
ST23/ClusterA3
Y:14:NST
7-’06
55
Septicaemia
3171
ST23/ClusterA3
Y:NT:P1.5
7-’06
46
Meningitis and Septicaemia
3171
ST23/ClusterA3
Y:14:NST
8-’06
7
Meningitis and Septicaemia
3171
ST23/ClusterA3
Y:NT:P1.5,2
12-’06
18
Meningitis
3171
ST23/ClusterA3
1n.a.:
0
0.023
0.032
0.047
0.064
0.094
0 , 12 5
0 , 19
Breakpoint for PenI
Sequence analysis of the 402 bp region of the penA gene showed the presence of 3
alleles: the alleles 20 , 9 and 22 (Figure 3)
0.05
Y_14NST
Figure 3. Phylogenetic tree based on 402 bp
alignment of C-terminal part of penA gene
sequences
of
PenI
serogroup-Y
Neisseria
meningitidis
allele 20
Y4.14_NST_N=2
Y14NST
allele 9
Y_NT_P1.5_2
allele 22
not available
Table 1. Phenotypic and genotypic characteristics of serogroup Y
meniongococci isolated in Italy from 2004 through 2006
The majority of the serogroup Y isolates belong to the ST-23complex/Cluster
A3, in particular 66.6% in 2004 and 100% in 2005 and 2006 (Figure 1)
Alleles 9 and 20 showed an identical amino acid sequence, with an identical mosaic
structure. On the contrary, the allele 22 differs from alleles 9 and 20 for 14 amino
acids and for the lack of mosaicism (Figure 4)
Allele 9
DGVLLPVSFEKQAVAPQGKRIFKASTARQVRELMVSVTEPGGTGTAGAVDGFDVGAKTGTARKLVNGRYVDYKHVATFIG
Allele 20
DGVLLPVSFEKQAVAPQGKRIFKASTARQVRELMVSVTEPGGTGTAGAVDGFDVGAKTGTARKLVNGRYVDYKHVATFIG
Allele 22
DGVLLPVSFEKQAVAPQGKRIFKESTAREVRNLMVSVTEPGGTGTAGAVDGFDVGAKTGTARK FVNGRYADNKHIATFIG
Allele 9
FAPAKNPRVIVAVTIDEPTANGYYGGVVTGPVFKQVMGGSLNILGVSPTKPLTN
Allele 20
FAPAKNPRVIVAVTIDEPTANGYYGGVVTGPVFKQVMGGSLNILGVSPTKPLTN
Allele 22
FAPAKNPRVIVAVTIDEPTAHGYYGGVVAGPPFKK IMGGSLNILGISPTKPLTA
8
Figure 1. Distribution of all serogroup Y
strains isolated from 2004 to 2006
(yellow bars) vs serogroup Y strains
belonging to ST-23complex/ClusterA3
(blue bars)
7
6
5
4
3
2
1
Figure
4.
Partial
sequences of the Cterminal part of PBP2
(AA 441-574) of PenI
strains serogroup Y
No association was detected between penA alleles and MIC values in PenI strains
0
2004
2005
2006
PFGE analysis showed the clonal relationship for 85% of ST-23/Cluster A3
strains. All clonal strains were phenotype Y:14:NST or Y:4,14:NST. Two
isolates, with phenotypes Y:NT:P1.5 and Y:NT:P1.5,2, were unrelated with
the main pulse type (data not shown).
Phenotype
penA allele
MIC (mg/L) for
penicillin G
Y:14:NST
9
0.19
Y:14:NST
20
0.125
Y:4,14:NST
20
0.125
Y:4,14:NST
20
0.094
Y:NT:P1.5,2
22
0.125
Table 2
CONCLUSIONS
This study describes the phenotypic
and genotypic characteristics of all
Neisseria meningitidis serogroup Y
strains isolated from recent cases of
meningitis and septicaemia in Italy
The majority of the isolates
belong
to
the
ST-23
complex/Cluster A3, already
described as responsible for
meningococcal
disease
worldwide
Although serogroup Y meningococci
are still an uncommon cause of
invasive disease in the country, the
number of isolates belonging to this
particular phenotype and genotype
increased during 2006
All the meningococci serogroup Y with
decreased
susceptibility
to
penicillin
belong to ST23/A3 complex. All of them,
except one, were characterized by the
presence of a mosaic structure in the
penA gene.
This work was partially supported by the Ministry of Health-CCM grant n°7M21 “Surveillance of Invasive Bacterial Infections” and FIRB project NIC of the Italian Ministry of Universities and Research