Characterisation of invasive meningococcal isolates from Italian children and adolescents

100 Clinical Microbiology and Infection, Volume 13 Number 1, January 2007

11. Yokota M, Ichihara S, Lin TL, Nakashima N, Yamada Y. Association of a T29C polymorphism of the transforming growth factor-b1 gene with genetic susceptibility to myocardial infarction in Japanese. Circulation 2000; 101: 2783– 2787. 12. McGuirk P, Mills KHG. Pathogen-specific regulatory T cells provoke a shift in the Th1 ⁄ Th2 paradigm in immunity to infectious diseases. Trends Immunol 2002; 23: 450–455. 13. Redpath S, Ghazal P, Gascoigne NR. Hijacking and exploitation of IL-10 by intracellular pathogens. Trends Immunol 2001; 9: 86–92. 14. Kehrl JH. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986; 163: 1037–1050. 15. Swain SL, Huston G, Tonkonogy S, Weinberg A. Transforming growth factor-beta and IL-4 cause helper T cell precursors to develop into distinct effector helper cells that differ in lymphokine secretion pattern and cell surface phenotype. J Immunol 1991; 147: 2991–3000. 16. Artavais-Tsakonas K, Tongren JE, Riley EM. The war between the malaria parasite and immune system; immunity, immunoregulation and immunopathology. Clin Exp Immunol 2003; 133: 145–152.

RESEARCH NOTE Characterisation of invasive meningococcal isolates from Italian children and adolescents P. Mastrantonio, T. Sofia, A. Neri, C. Fazio and P. Stefanelli Department of Infectious, Parasitic and ImmuneMediated Diseases, Istituto Superiore di Sanita` (ISS), Rome, Italy

ABSTRACT Meningococcal invasive disease is a life-threatening infection that affects mostly children and adolescents. The present study was performed during 2003–2005 to compare the phenotypic characteristics of meningococcal isolates from these two main groups at risk with those of isolates from other age groups to assess whether strategies for treatment and prevention impleCorresponding author and reprint requests: P. Mastrantonio, Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanita`,Viale Regina Elena 299, 00161 Rome, Italy E-mail: [email protected]

mented elsewhere can also be applied in Italy. The results showed that serogroup C meningococci were predominant, and that a dramatic increase in the circulation of strains with decreased susceptibility to penicillin was associated mainly with a prevalent phenotype C:2b:P1.5,2, which belongs to the hyper-virulent ST8 ⁄ A4 cluster. Keywords Adolescents, children, group C meningococci, Italy, meningitis, Neisseria meningitidis Original Submission: 19 April 2006; Revised Submission: 19 June 2006; Accepted: 5 July 2006

Clin Microbiol Infect 2007; 13: 100–103 10.1111/j.1469-0691.2006.01570.x Children in the first few years of life and adolescents are the two age groups most at risk of developing invasive meningococcal disease. Despite the availability of a targeted therapy, the disease remains life-threatening, and many efforts have been made in recent years to design safe immunogenic vaccines. Currently, polysaccharide and conjugate vaccines can prevent cases caused by Neiserria meningitidis serogroups A, C, Y and W135, and serogroup C only, respectively [1,2]. In addition, the newer outer-membrane vesicle (OMV) vaccines, which contain the meningococcal outer-membrane proteins (PorA) of class 1 that are normally used for strain subtyping [3], seem to be able to overcome the disadvantages of immunogenicity and cross-reactivity with host tissue that occur with the meningococcal B polysaccharide [4]. The main constraint in the development of OMV vaccines is the great diversity of sero-subtypes circulating in different geographical areas, which would require ad-hoc vaccine production tailored to each specific situation [5,6]. Meningococci with decreased susceptibility to penicillin (PenI) have been described since the mid-1980s [7], and their frequency is increasing constantly throughout the world. Although fullyresistant strains have not yet been isolated in Italy, the situation requires constant monitoring. The aim of the present work was to assess the meningococcal phenotypes circulating during 2003–2005 in Italy in the two groups most at risk, i.e., children aged £4 years and adolescents aged 15–19 years, since different opportunities are available for prophylaxis and it is imperative for national public health laboratories to have

 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 86–108

Research Notes 101

up-to-date data concerning the phenotypic characteristics of meningococci isolated from cases of invasive disease. National surveillance of meningococcal invasive disease is ongoing in Italy, and the Istituto Superiore di Sanita`, Rome acts as a national reference laboratory, with data being forwarded to the EU Invasive Bacterial Infection Surveillance Network (EU-IBIS, http:// www.euibis.org) every 6 months. During 2003–2005, 923 cases of invasive meningococcal disease were notified to the national surveillance network. Of these, 670 (72%) were confirmed by culture; the National Reference Laboratory received 433 viable isolates, mainly from cerebrospinal fluid or blood of patients. The isolates were subcultured on Thayer Martin agar, and the serogroup was then identified by slide agglutination with commercial antisera (Oxoid, Milan, Italy). Serotyping and subtyping were performed by whole-cell enzyme-linked immunoassay with monoclonal antibodies (NIBSC, Potters Bar, UK) [8]. MICs of penicillin, rifampicin, ceftriaxone and sulphadiazine were determined by Etest (AB Biodisk, Solna, Sweden) on Mueller– Hinton agar (Oxoid), supplemented with lysed horse blood 5% v ⁄ v, incubated in CO2 5% at 35C for 24 h. Breakpoint criteria were those recommended by the CLSI [9]. Of the 433 viable isolates, 227 were serogroup C, 188 were serogroup B, eight were serogroup Y, seven were serogroup W135, and three were nonserogroupable. Serotype and subtype could be determined for 76% of the isolates, but 38 isolates were not serotypeable, 50 were not subtypeable, and 15 were neither serotypeable nor subtypeable. Serogroup C was found to have replaced serogroup B as the predominant serogroup since

Table 1. Serogroup B and serogroup C meningococci circulating in Italy during 2003–2005

2004. The most common phenotypes during the 3year period were C:2b:P1.5,2 (22.3%), C:2b:P1.5 (7.8%) and B:15:P1.4 (7%) (Table 1). The prevalent phenotypes of isolates from children and adolescents reflected the trend described above, with 117 serogroup C isolates representing 61% of all meningococci isolated from these two age groups. Overall, the C:2b:P1.5,2 phenotype was predominant, accounting for 50% of isolates in children and 41% in adolescents. Serogroup B accounted for just over one-third of meningococci detected in these two age groups, but no particular phenotype was predominant, with B:15:P1.4 and B:4:P1.3,6 each representing c. 10% of isolates. Examining the changes in the most prevalent phenotypes within each serogroup on an annual basis, phenotype C:2b:P1.5 disappeared in 2005, although it accounted for 46.3% of isolates during 2003. The C:2b:P1.5,2 phenotype appeared for the first time in 2003, and then increased to account for 50% and 59.3% of isolates in 2004 and 2005, respectively. Selected isolates from these two phenotypes that were examined by multilocus sequence typing [10] were all assigned to the ST8 ⁄ A4 cluster. A wide variety of sero ⁄ subtypes was observed among serogroup B isolates, but some minor changes occurred. Thus, B:15:P.1.4 accounted for 10.1% of isolates in 2005, compared with 18.6% and 20% of isolates in 2003 and 2004, respectively, and B:NT:P1.9 accounted for 1.4% of isolates in 2003, compared with 7.2% of isolates in 2005. The relative proportions of other phenotypes, e.g., B:14:P1.13, B:15:P1.7,16 and B:4:P1.4, did not vary significantly, accounting for 4–7% of isolates. Non-serotypeable isolates were more frequent among serogroup B isolates (17.4% in

Serogroup B

Serogroup C

Phenotype

2003

2004

2005

Phenotype

2003

2004

2005

B:14:P1.13 B:15:P1.16 B:15:P1.4 B:15:P1.7,16 B:4:P1.13 B:4:P1.15 B:4:P1.3,6 B:4:P1.4 B:nt:nst B:nt:P1.5 B:nt:P1.9 Other phenotypes Total

5 5 13 4 2 2 2 4 3 4 1 23

2 2 10 2 7 2 2 2 2 0 4 15

4 2 7 4 4 4 5 3 3 0 5 29

C:2a:P1.5 C:2b:nst C:2b:P1.2 C:2b:P1.5 C:2b:P1.5,2 C:nt:nst

10 5 0 25 2 4

4 12 4 9 41 1

9 8 5 0 54 1

Other phenotypes

8

11

14

68

50

70

54

82

91

nt, not typeable; nst, not subtypeable.

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102 Clinical Microbiology and Infection, Volume 13 Number 1, January 2007

Table 2. Antimicrobial suceptibility data for 433 meningococci circulating in Italy during 2003–2005 2003

2004

2005

Antibiotic

MIC50

MIC90

Range

MIC50

MIC90

Range

MIC50

MIC90

Range

Penicillin Serogroup B isolates Serogroup C isolates Rifampicin Sulphadiazine Ceftriaxone

0.047 0.094 0.016 >256 <0.002

0.064 0.190 0.064 >256 <0.002

0.012–0.380 0.012–0.380 <0.002–0.250 0.064 to >256 <0.002–0.023

0.032 0.125 0.016 >256 <0.002

0.094 0.250 0.064 >256 <0.002

0.012–0.250 0.023–0.380 <0.002–256 to >256 0.064–256 <0.002–0.004

0.064 0.125 0.016 >256 <0.002

0.125 0.190 0.032 >256 <0.002

0.012–0.250 0.023–0.180 <0.002–0.380 0.125 to >256 <0.002–0.094

MIC values are expressed in mg ⁄ L.

2005) than among serogroup C isolates (6.6%), while non-subtypeable isolates were distributed equally (13%) between the two serogroups. Table 2 shows MIC50 and MIC90 values of penicillin, rifampicin, sulphadiazine and ceftriaxone. Decreased susceptibility to penicillin increased among serogroup C isolates during the 3-year period, from 50.9% to 87.4%. This was associated mainly with C:2b:P1.5,2 isolates; since the first appearance of this phenotype in 2003, >95% of isolates have shown decreased susceptibility to penicillin (MICs 0.094–0.380 mg ⁄ L). Since this phenotype was predominant in all age groups, no great difference was observed in terms of distribution of serogroup C PenI isolates among the different age groups. In contrast, a greater proportion of serogroup B isolates with decreased susceptibility to penicillin was isolated from children and adolescents than from other age groups. Interestingly, resistance to rifampicin was rare [11] (only one isolate in 2004 from a child aged 4 years), despite its long use for prophylaxis of close contacts, and sometimes for entire small communities, e.g., schools, colleges and gymnasiums. In conclusion, serogroup C meningococci were predominant in the two age groups most at risk of developing disease during 2003–2005 in Italy. The dramatic increase in the circulation of meningococci with decreased susceptibility to penicillin requires careful monitoring, and this issue must be considered when determining vaccination strategies. Serogroup B isolates showed a large range of different phenotypes and therefore the use of OMV vaccines does not seem to be feasible at the present time. Indeed, none of the subtypes included in the hexavalent OMV vaccine that is currently being evaluated in the UK (i.e., P1.7,16; P1.5,2; P1.19,15; P1.7; P1.5; and P1.12,13) [12] was significantly represented among group B isolates circulating in Italy.

ACKNOWLEDGEMENTS We thank the personnel of all the microbiology laboratories participating in the Italian National Surveillance of Bacterial Meningitis for obtaining the isolates and sending them to the reference laboratory at the Istituto Superiore di Sanita`. This study was partially funded by the Ministry of Health-CCM project N17, ‘Diseases Preventable Through Vaccination’.

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Research Notes 103

11. Stefanelli P, Fazio C, La Rosa G, Marianelli C, Muscillo M, Mastrantonio P. Rifampicin-resistant meningococci causing invasive disease: detection of point mutations in the rpoB gene and molecular characterization of the strains. J Antimicrob Chemother 2001; 47: 219–222. 12. Findlow J, Lowe A, Deane S et al. Effect of sequence variation in meningococcal PorA outer membrane protein on the effectiveness of a hexavalent PorA outer membrane vescicle vaccine in toddlers and school children. Vaccine 2005; 23: 2623–2627.

RESEARCH NOTE Prevalence and mechanisms of macrolide resistance among Staphylococcus epidermidis isolates from neutropenic patients in Tunisia O. Bouchami, W. Achour and A. Ben Hassen National Bone Marrow Transplant Centre, Tunis, Tunisia

ABSTRACT The prevalence of macrolide–lincosamide–streptogramin (MLS) resistance phenotypes was determined among erythromycin-resistant Staphylococcus epidermidis isolates collected at the Bone Marrow Transplant Centre, Tunisia during 2002. The erm(A), erm(B), erm(C), msrA, mefA and icaA genes were detected by PCR. The vga, vgb and vat genes were amplified from pristinamycinresistant isolates. The icaA gene was detected in 76.5% of 34 isolates examined in detail. The erm(C) (53%) and erm(A) (32%) genes predominated because of clonal dissemination, followed by msrA (15%). Gene distribution was related to the methicillin resistance pattern. The vga gene was present in combination with erm(A) in three isolates. Keywords Coagulase-negative staphylococci, icaA gene, macrolide resistance genes, resistance, Staphylococcus epidermidis, Tunisia

Corresponding author and reprint requests: A. Ben Hasen, Laboratory, National Bone Marrow Transplant Centre, Tunis, Tunisia E-mail: [email protected]

Original Submission: 8 June 2005; Revised Submission: 31 May 2006; Accepted: 3 July 2006

Clin Microbiol Infect 2007; 13: 103–106 10.1111/j.1469-0691.2006.01567.x Coagulase-negative staphylococci are the bacteria isolated most frequently from the blood cultures of febrile neutropenic patients, particularly in association with the use of intravenous catheters [1]. Resistance to macrolides is conferred either by methylases, encoded by erm genes that modify the ribosomal target of macrolides [2], or by efflux pumps, encoded by the msrA gene. Ribosomal methylation results in cross-resistance to macrolide, lincosamide and streptogramin B antibiotics (MLSB phenotype) [3], and can be expressed either constitutively (cMLSB) or inducibly (iMLSB) [4]. Macrolide efflux is characterised by resistance to 14- and 15-membered-ring macrolides (M phenotype) [5]. The aims of the present study were to determine the incidence and patterns of antimicrobial resistance among Staphylococcus epidermidis isolates from Tunisia, to correlate erythromycin and pristinamycin resistance with the presence of the MLS resistance genes erm(A), erm(B), erm(C), msrA, mefA, vga, vgb and vat, to search for the presence of the icaA gene in these isolates, and to assess their molecular epidemiology. During 2002, 55 S. epidermidis isolates from blood cultures and 22 isolates from the intravascular catheters of 34 patients with haematological cancer at the Centre National de Greffe de Moelle Osseuse, Tunis, Tunisia were analysed. Isolates were identified using conventional methods and the ID-32 Staph system (bioMe´rieux, Marcy l’Etoile, France). Susceptibility to antibiotics was tested using the agar diffusion technique. MICs were determined using the agar dilution method according to French guidelines [6]. Resistance phenotypes of erythromycin-resistant isolates were determined using erythromycin, spiramycin, clindamycin, lincomycin and pristinamycin disks. Thirty-four erythromycin-resistant isolates were chosen randomly for PCR amplification of macrolide resistance genes [7–9] using the primers shown in Table 1. A multiplex PCR assay was used to amplify erm(A), erm(C) and msrA [8]; a duplex PCR was used to amplify erm(B) and mefA [10]; and individual PCRs were used to detect vga, vgb, vat [11] and icaA [9]. Pulsed-field gel

 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 86–108