Clonal structure and 21-year evolution of Streptococcus pneumoniae serotype 1 isolates in northern Spain

Clonal structure and 21-year evolution of Streptococcus pneumoniae serotype 1 isolates in northern Spain

RESEARCH NOTES Clonal structure and 21-year evolution of Streptococcus pneumoniae serotype 1 isolates in northern Spain J. M. Marimon1,2, M. Ercibeng...

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RESEARCH NOTES

Clonal structure and 21-year evolution of Streptococcus pneumoniae serotype 1 isolates in northern Spain J. M. Marimon1,2, M. Ercibengoa2, M. Alonso 1

1,2

,

1,2,3

M. Zubizarreta and E. Pe´rez-Trallero 1) Servicio de Microbiologı´a, Hospital Donostia, 2) Centro de Investigacio´n Biome´dica en Red Enfermedades Respiratorias CIBERES and 3) Departamento de Medicina Preventiva y Salud Pu´blica, Facultad de Medicina, Universidad del Paı´s Vasco, San Sebastia´n, Spain

Abstract This study of 135 serotype 1 pneumococcal isolates (88 invasive and 47 non-invasive), collected between 1987 and 2007, gave eight sequence types (217, 227, 228, 304, 305, 306, 3860 and 3861) that group, using eBurst, into three different lineages and one singleton. The annual incidence of serotype 1 invasive episodes per million inhabitants increased from 1.8 in 1987–1993 to 4.0 in 1994–2000, and to 25.6 in 2001–2007. ST228 was the predominant clone until 1998. ST306 first appeared in 1998 and became the most prevalent sequence type (>80%) after the introduction, in June 2001, of the heptavalent pneumococcal conjugate vaccine.

Keywords: Lineages, MLST, PCV7, pneumococcal conjugate vaccine, pneumococcal infection Original Submission: 20 October 2008; Revised Submission: 3 March 2009; Acceptance: 4 March 2009 Editor: P Tassios Clin Microbiol Infect 2009; 15: 875–877 10.1111/j.1469-0691.2009.02883.x

Corresponding author and reprint requests: E. Pe´rez-Trallero, Servicio de Microbiologı´a, Hospital Donostia, Paseo Dr. Beguiristain s/n, 20014 San Sebastia´n, Spain E-mail: [email protected]

Streptococcus pneumoniae serotype 1 is considered a primary pathogen, since it is more frequently isolated from sterile locations than from non-sterile samples or carriers [1]. Indeed, serotype 1 has been associated with outbreaks of invasive disease [2]. At the beginning of the 21st century, the 7-valent pneumococcal conjugate vaccine (PCV7) was

introduced for children. Vaccination has changed the distribution of serotypes causing invasive pneumococcal disease in children, substantially decreasing the incidence of vaccine serotypes (serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and increasing some of the serotypes not included in this vaccine [3–5]. The objective of this study was to determine the clonal structure and evolution of serotype 1 isolates causing invasive and non-invasive disease in a region of northern Spain in the last 21 years. This study was performed in the region of Gipuzkoa, Basque Country, northern Spain, a province bordered by the Bay of Biscay and France. This region includes the areas of San Sebastia´n, Tolosa and most of the villages belonging to the Urola Coastal area. Population data were based on the official censuses for 1991, 1996, 2001 and 2006 (Eustat, http://www.eustat.es). The PCV7 was introduced in Spain in June 2001 and approximately 50% of children aged <2 years have been vaccinated in the study region [6]. Overall, 135 non-duplicated serotype 1 isolates were studied. Between 1987 and 2007, 88 (7.28%) out of 1209 pneumococcal isolates causing invasive disease in the population-based area of the study were serotype 1. The remaining isolates were: 24 from the ear fluid of children with otitis and 23 from the sputum of adults with respiratory infection (Table 1). The identification, serotyping and genotyping by pulsed field gel electrophoresis (PFGE) were performed according to previously described standard protocols [7]. Antimicrobial susceptibility [8] and multilocus sequence typing (MLST) [9] were performed as described previously. MLST was performed on all isolates and yielded eight different sequence types (STs) (http://spneumoniae.mlst.net/ eburst/). The 135 isolates were grouped by eBURSTv3 into three lineages, arbitrarily named I to III, and, if only serotype I isolates included in the MLST database were considered, one singleton, ST3860 (Table 2). According to eBURST, the STs in each lineage were evolutionarily related. MLST-lineage-I included most of the serotype 1 isolates (123/135, 91.1%). The singleton is a newly described ST3860 (single locus variant of ST1340), probably resulting from genetic switching of a capsular type 19F isolate, since ST3860 was the only component of this lineage that was not serotype 19F. The eight different MLST-types coincided with the PFGE patterns when the latter were defined by a similarity of ‡85% (Table 2). When a PFGE similarity of between 85% and <95% was considered in the clustering analysis of type A isolates, these could be further divided into six subtypes (arbitrarily named A1–A6) (Table 2). All 135 serotype 1 isolates were penicillin-susceptible (MIC <0.12 mg/L), rifampicin-susceptible (MIC <2 mg/L) and erythromycin-susceptible (MIC <0.50 mg/L); one was

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TABLE 1. Temporal distribution of Streptococcus pneumoniae serotype 1 isolates in Gipuzkoa, northern Spain, from 1987 to 2007 according to sequence types (ST) Invasive isolates from the population-based area (incidence per million inhabitants) Overall

1987–1993

5

1994–2000

(1.8) 11

2001–2007

(4.0) 72

<5 years

1 ST228 1 ST304 (18.6) 10 ST306

5–14 years

1 ST306 (4.2) 7 ST306 1 ST228

(25.6)a 88

Total

(34.7)a 9

(78.0) 12

Non-invasive isolates

15–64 years

>64-years

Otic

Sputum

2 ST228 1 ST305 1 ST3860 (2.0) 3 ST228 3 ST306 (3.0) 24 ST306 5 ST228 2 ST227 1 ST304 1 ST217 (16.7)a 43

1 ST304

4 ST304

1 ST3861

3 ST228 1 ST306

2 ST228 1 ST306

(4.6) 13 ST306 6 ST228 1 ST227 1 ST305

11 ST306 4 ST227 1 ST228

17 ST306 1 ST228 1 ST227

(44.0)a 24

24

23

(2.8) 2 ST228

a

Significant increase in the incidence of each previous period and the 2001–2007 period. No significant increase was observed in the incidence between the 1987–1993 and the 1994–2000 periods.

TABLE 2. Lineages and STs of Streptococcus pneumoniae serotype1 isolates from Gipuzkoa, found between 1987 and 2007 Allelic profile Lineage

ST

No. of isolates

PFGE patterns

Years of isolation

aroE

gdh

gki

recP

spi

xpt

ddl

I

227 228 228 306 306 306 304 3861a 305 217 3860b

8 24 1 81 7 1 7 1 2 1 1

A5 A1 A6 A2 A3 A4 B B B C D

2002–2006 1993–2007 1994 1998–2007 2004–2006 2006 1990–2004 1990 1993–2002 2007 1989

12 12 12 12 12 12 13 13 13 10 10

5 8 8 8 8 8 8 10 8 18 5

13 1 1 13 13 13 13 13 13 4 4

5 5 5 5 5 5 5 5 5 1 1

17 17 17 16 16 16 17 17 17 7 6

4 4 4 4 4 4 4 4 4 19 4

20 20 20 20 20 20 8 8 28 9 8

II

III Singleton

PFGE, pulsed-field gel electrophoresis; ST, sequence type. New ST. New ST. Typical of serotype 19F.

a

b

trimethoprim-sulfamethoxazole-resistant (MIC >4/76), three were ciprofloxacin-resistant (MIC ‡4 mg/L), and two tetracycline-resistant (MIC ‡4 mg/L). The absolute number and incidence of invasive serotype 1 isolates increased throughout the period of study (Table 1). This increase was non-significant between 1987–1993 and 1994–2000, but significant between each of the subsequent periods and 2001–2007 (p <0.0001). The difference remained significant when comparing each age group separately, except for the under-five age group. In this age group, perhaps due to the low number of cases, significance was evident only when comparing the first and last periods (chi-square with Yates correction v2 Yates = 5.53, p 0.019). Because the PCV7 vaccine was introduced in June 2001, the incidence rates for serotype 1 invasive infections between the prevaccination (January 1987 to June 2001) and post-vaccination periods (July 2001 to December 2007) were compared; significant increases were observed in the overall population

(v2 Yates = 53.94, p <0.0001) and in the population groups of 5–14 years (v2 Yates = 5.29, p 0.02), 15–65 years (v2 Yates = 22.19, p >0.0001) and >64 years (v2 Yates = 15.16, p >0.0001). The increasing incidence in the population aged <5 years was non-significant (v2 Yates = 3.49, p 0.062). Within the total number of invasive isolates, serotype I increased through the study period from 2.4% (5/207) in 1987–1993 and 2.7% (11/405) in 1994–2000, to 12.1% (72/597) in 2001–2007. Between 1987 and 1995, serotype 1 invasive isolates were isolated only from adults; by the end of the 1990s, however, serotype 1 was frequent among invasive isolates from all age groups, at a frequency that markedly increased in young children compared with adults between 2005 and 2007. After the introduction of the PCV7, nine children aged <5 years with serotype 1 invasive disease had pneumonia, accompanied by pleural effusion in three cases. Seven of these children had received at least one dose of PCV7.

ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 875–884

Research Notes

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Between 1987 and 1998 the distribution of clones showed wide heterogeneity. ST228 was the predominant clone among all serotype 1 isolates, representing 61.5% (8/13) of those causing invasive disease, but its incidence subsequently decreased to 13.3% (10/75) after 1999 (p <0.001). ST306 appeared for the first time in 1998 in the blood culture of a 29-year-old man with pneumonia, and subsequently became dominant: between 1998 and 2007, ST306 comprised 58/78 (74.4%) of the invasive isolates and 30/39 (76.9%) of noninvasive isolates. Some authors have suggested an association between the increase in the number of invasive pneumococcal disease cases caused by serotype 1 and the use of the PCV7 [10– 12]. In our study region, a wide circulation of serotype 1 isolates was observed before the introduction of this vaccine, but a rapid increase in its incidence was observed after the introduction of the PCV7, with ST306 being the most prevalent sequence-type. This study corroborates findings in the rest of Europe and North America that serotype 1 is associated with ST306 and its related sequence types from the same lineage [1,13–15] and can therefore be considered to be a clone or group of clones whose current behaviour is near pandemic.

Acknowledgement We acknowledge the use of the pneumococcal MLST database, which is located at Imperial College London and is funded by the Wellcome Trust.

Transparency Declaration The authors declare no conflict of interest in relation to this study.

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ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 875–884