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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