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Journal of Clinical Microbiology, November 2005, p. 5665-5669, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5665-5669.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characteristics of Haemophilus influenzae Type b Responsible for Meningitis in Poland from 1997 to 2004

Anna Skoczyska,^1* Marcin Kadubowski,¹ Joanna Empel,² and Waleria Hryniewicz¹

National Reference Centre for Bacterial Meningitis, Department of Epidemiology and Clinical Microbiology,¹ Department of Molecular Microbiology, National Institute of Public Health, Warsaw, Poland²

Received 13 July 2005/ Returned for modification 1 August 2005/ Accepted 6 September 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two hundred forty-five H. influenzae isolates responsible for meningitis in Poland from 1997 to 2004 were studied. Among these, 233 (95.1%) belonged to serotype b (Hib), 2 belonged to serotype f, and 10 were noncapsulated. The relatedness of all isolates was evaluated by pulsed-field gel electrophoresis (PFGE), and selected representatives were evaluated by multilocus sequence typing. Resistance to ampicillin was identified in 34 (14.6%) of the Hib isolates and was associated with the production of ß-lactamase only. Except for four isolates nonsusceptible to chloramphenicol, all isolates were susceptible to cefotaxime, ciprofloxacin, and rifampin. The PFGE analysis divided the Hib isolates into five PFGE types; however, all of them were possibly related. The most common PFGE type, with 25 subtypes, was characteristic for 97.4% of the isolates. The most prevalent PFGE subtype found in our study was also the most common among the Hib isolates responsible for invasive disease in Italy and the Czech Republic and was found among isolates causing lower respiratory tract infections in Poland. The most prevalent sequence types (STs) in the studied group were ST6 and ST92. Four new STs were found: ST188, ST189, ST190, and ST268. Results of this study support the evidence that the genetic structure of encapsulated H. influenzae is clonal. The continuing high number of meningitis cases due to Hib in Poland underlines the need for mass vaccination against Hib in Poland.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In many countries with no mass vaccination program Haemophilus influenzae is still one of the most common etiologic agents of acute bacterial meningitis, mainly in children under 5 years of age. In addition to meningitis, this pathogen is responsible for other severe invasive infections, such as bacteremia/sepsis, epiglottitis, and septic arthritis, as well as for upper and lower respiratory tract infections. Isolates of this species, found exclusively in humans, can be encapsulated or noncapsulated (nontypeable). Nontypeable isolates can be carried by up to 80% of healthy people. Encapsulated isolates, which are less frequent, are divided into six serotypes, from a to f, based on their antigenic and structural diversity (20). All over the world the majority of severe infections related to this species are caused by H. influenzae type b (Hib). In countries where mass vaccination against Hib has been introduced, a dramatic reduction in the number of cases, as well as a noticeable decline of carriage rate in the population, has been reported (20, 24, 25, 27). Although highly recommended, vaccination against Hib in Poland is on a voluntary basis and not free of charge. After Neisseria meningitidis, therefore, Hib is still the second most common etiologic agent of bacterial meningitis in our country. Presently, in Poland, the vaccine is offered without charge only for children at the highest risk of contracting Hib infections, i.e., children in orphanages (since 2004) and children from families with at least three children under 5 years of age (since 2005) (35). Thus, the aim of this study was to investigate the molecular epidemiology and susceptibility to antibiotics of Haemophilus influenzae type b isolates responsible for meningitis in Poland.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. The laboratory-based surveillance of bacterial meningitis started in Poland in 1997, from the beginning of the activity of the National Reference Centre for Bacterial Meningitis (NRCBM). Between January 1997 and December 2004, the NRCBM collected 245 H. influenzae isolates from cerebrospinal fluid (CSF) or from blood when clinical symptoms of meningitis were observed despite negative results of the CSF culture. They constituted from 26.7% (in 1998) to 61.0% (in 2003) of notified cases, with an average of 40.1% (22). For comparison, the study also included representatives of Hib isolates of the most prevalent pulsed-field gel electrophoresis (PFGE) types responsible for invasive disease in Italy and the Czech Republic (28, 32). All isolates were identified to the species level based on standard procedures (14). The present investigation includes H. influenzae isolates from 1997 to 1998, described in a previous study; however, they were only characterized phenotypically (29).

The quality control strains of H. influenzae of serotypes a to f used for PCR were provided by the Oxford Public Health Laboratory (John Radcliffe Hospital, Oxford, United Kingdom). The Haemophilus parainfluenzae strain used as a negative control was derived from our collection (MIKROBANK reference no. 2259/96). The quality control strains for antimicrobial susceptibility testing used during this study were H. influenzae ATCC 49247 and H. influenzae ATCC 10211.

Susceptibility testing. MICs were determined by the broth microdilution method and interpreted according to CLSI guidelines (5). The following antimicrobial agents were tested: ampicillin (Polfa Tarchomin, Warsaw, Poland), rifampin (Polfa Tarchomin), cefotaxime (Roussel, Poteaux, France), ciprofloxacin (Bayer, Wuppertal, Germany), and chloramphenicol (Boehringer Mannheim, Mannheim, Germany) on Haemophilus test medium made in-house. The production of ß-lactamases was determined by the nitrocefin assay according to the manufacturer's instructions (Becton Dickinson, Meylan, France).

DNA preparation and PCRs. Species identification, serotype determination, and detection of capsule-specific genes were confirmed by the modified PCR-based methods previously described (7, 9). Eight to 12 colonies of H. influenzae isolates were suspended in 200 µl of distilled water, boiled for 10 min, and centrifuged at 12,000 x g for 5 min. Obtained supernatants were used for PCRs. For species identification primers O₁ and O₃ (9) were used, and for capsule gene detection primers HI-1 and HI-2 (7) were used. The PCRs for serotype a to f determination were run with primers a1-a2, b1-b2, c1-c2, d1-d2, e1-e2, and f1-f2, respectively (7). Reactions were run under the following conditions: 5 min at 94°C, followed by 25 cycles of 15 s at 94°C, 15 s at 55°C, and 30 s at 72°C, and finally 7 min at 72°C.

Preparation of genomic DNA and pulsed-field gel electrophoresis. The relatedness among encapsulated isolates was evaluated by restriction fragment length polymorphism of SmaI-digested chromosomal DNA following PFGE, using a method described previously by Tarasi et al. (32). The isolates were classified into PFGE types according to the interpretive criteria proposed by Tenover et al. (33). PFGE types representing identical PFGE patterns were designated by the same letter and Arabic number (indistinguishable isolates). Patterns with no more than a three band difference (closely related) were designated by the same letter and different Arabic numbers (the same PFGE type, different subtype). PFGE patterns showing four to six band differences were named as different PFGE types (different letters) but were reported as possibly related. PFGE patterns with more than a six band difference were labeled by different letters.

MLST. A multilocus sequence typing (MLST) technique, employed for the first time in Poland for H. influenzae typing, was carried out as described by Meats et al. (18). Alleles and sequence types (STs) were assigned using the MLST database (www.mlst.net).

Top Abstract Introduction Materials and Methods Results Discussion References

 
During the 8-year study period, 245 H. influenzae isolates responsible for meningitis were collected from 64 centers in Poland, representing the whole country (Fig. 1). The number of isolates obtained per year fluctuated from 24 in 1999 to 36 in 2003. Among the isolates studied, 233 (95.1%) belonged to serotype b (Hib) and two to serotype f (Hif). The remaining 10 isolates were noncapsulated. Of these, four were isolated in 2004. Among the Hib isolates, 214 were recovered from CSF and 19 from the blood of patients with clinical symptoms of meningitis. Although meningitis caused by Hib predominated in the age group of 6 to 24 months (n = 127; 54.5%), 12.4% (n = 29) of the patients with Hib meningitis were older than 5 years (27 cases affected patients 6 to 16 years old; 2 cases affected adults aged 65 and 69) and 3.4% (n = 8) were younger than 6 months. In seven cases, the patient's age was unknown.

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FIG. 1. Map of Poland highlighting the centers which provided Haemophilus influenzae isolates responsible for meningitis in Poland from 1997 to 2004.

Resistance to ampicillin was identified in 34 (14.6%) of the Hib isolates, and it was associated with the production of ß-lactamase. Nonsusceptibility to chloramphenicol was found exclusively among four isolates (three were fully resistant). These were also resistant to ampicillin. All isolates were susceptible to cefotaxime, ciprofloxacin, and rifampin. The MICs at which 50% of the isolates were inhibited (MIC₅₀s) and MIC₉₀s for ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, and rifampin were 0.25 and 4.0, 0.015 and 0.015, 0.5 and 1.0, 0.0075 and 0.03, and 0.25 and 0.5 µg/ml, respectively. The isolates of serotype f, as well as the nontypeable isolates, were susceptible to all the antimicrobials tested.

The PFGE analysis divided Hib isolates into five PFGE types. However, all of them were possibly related according to the criteria of Tenover et al. (33). The most common PFGE type, A, with 25 subtypes, was characteristic for 97.4% of the isolates. Among them, the most prevalent PFGE subtypes, A1, A13, A17, and A2, represented 55.1%, 15.4%, 6.6%, and 6.2% of the isolates, respectively. The representatives of Hib isolates of the most prevalent PFGE types responsible for invasive disease in Italy and the Czech Republic used in this study also belonged to PFGE subtype A1. Fourteen subtypes of PFGE type A were represented by one isolate only. Results of the PFGE analysis are presented in Table 1 and Fig. 2. All but one isolate resistant to ampicillin belonged to nine PFGE subtypes of type A. Twelve out of 15 isolates of PFGE type A17 were resistant to ampicillin. All isolates of PFGE subtypes A3, A4, A5, A6, A14, and A25 were found to produce ß-lactamases. Two out of 125, 1 out of 2, and 3 out of 5 isolates of PFGE subtypes A1, C, and A15, respectively, were also resistant to ampicillin.

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TABLE 1. Characteristics of encapsulated H. influenzae isolates responsible for meningitis in Poland from 1997 to 2004

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FIG. 2. Restriction fragment length polymorphism-PFGE patterns of encapsulated H. influenzae DNA digested with SmaI. Lanes contain molecular weight markers (Lambda DNA ladder; New England Biolabs, Beverly, Mass.). a, PFGE pattern of isolate from Italy; b, PFGE pattern of isolate from the Czech Republic.

MLST analysis was performed on 36 selected isolates, of which 31 corresponded to a randomly chosen member of each PFGE type/subtype. The remaining four represented ampicillin-susceptible and -resistant phenotypes when present within the same PFGE subtype/type. One exception was PFGE subtype A13, of which two susceptible isolates were tested. The most prevalent ST in the studied group was ST6, found in 17 subtypes of PFGE type A and 1 subtype of PFGE type B. The second most common ST was ST92, a single-locus variant (SLV) of ST6, characteristic for 5 PFGE subtypes of type A and PFGE type C. Additionally all isolates of PFGE types A, B, and C belonged to ST6; ST92; or ST44, -95, -189, -190, and -268. The last five differed from ST6 and ST92 by one or two loci. Four new STs were found during our study. Three of them, ST188, -189, and -190, were established as a result of new mdh alleles 83, 84, and 85, respectively. The new ST268 is an SLV of ST44 due to a new allele, 49, of the fucK gene. This allele differs from allele 3 of the fucK gene by a single adenine deletion in the tract of six consecutive adenines at positions 98 to 103. Among representatives of isolates resistant to ampicillin we found three STs, ST6, -92, and -190. Generally, the PFGE analysis was more discriminatory for isolates tested than was the MLST technique. However, in three cases, the MLST classified isolates of the same PFGE subtypes into diverse STs. Results of the MLST analysis are presented in Table 1.

Top Abstract Introduction Materials and Methods Results Discussion References

 
According to the Polish notification system, based mostly on a clinical picture of the disease, the annual incidence reported during the studied period fluctuated from 0.15 to 0.26 per 100,000 inhabitants for all ages (22). The population-based surveillance study conducted among children in two Polish districts, Kielce and Bydgoszcz, estimated the annual incidence of Hib meningitis to be 3.1 and 9.7 per 100,000 under 5 years of age, respectively (36). According to NRCBM laboratory-confirmed data, H. influenzae was the second most prevalent etiologic agent of bacterial meningitis in Poland in children under 5 years of age, following Neisseria meningitidis. The Polish incidence data differ significantly from, e.g., U.S., English and Welsh, or Slovakian data, where, before the introduction of mass Hib vaccination, the rates of Hib meningitis incidence were 50 to 60, 22, and 17.3 per 100,000 under 5 years of age, respectively (12, 23, 25). However, that is not always the case, since Japan, despite a lack of mass vaccination, reported an incidence of only 4.0 per 100,000 (19). Our study revealed that more than 95% of the Polish meningitis H. influenzae isolates belonged to serotype b. Such results are not surprising and are comparable to data from other countries which have not introduced a national vaccination program against Hib or to data from a prevaccination era (13, 20). Generally, as was expected, most infections in our study affected children 6 to 24 months old, but, interestingly, more than 12% of the patients were older than 5 years. These results resemble results found in England and Wales, where, in the prevaccination era, 12% of the cases affected patients older than 5 years (1).

During our study we found two isolates of serotype f which, of non-Hib serotypes, is known to be the most frequently associated with invasive disease. Additionally, it has been reported that the incidence of Hif disease in some vaccinated populations increased in recent years and that the mortality rate was very high, even up to 30% (18, 30). The first Hif isolate in our study, recovered in 2000, was initially identified by a latex agglutination test in a local laboratory as Hib. With the second one, from 2004, the situation was similar, but additionally the case was reported as a vaccine failure, since the patient had been vaccinated against Hib. The NRCBM retyped isolates by PCR, and correct serotypes were established. Therefore, it should be borne in mind, especially when Hib vaccine failures are encountered, that, because in some isolates expression of capsular polysaccharide is not sufficient to be detected by slide agglutination, the PCR technique should be used as a standard method in serotyping of H. influenzae (3, 16, 24). Moreover, the study performed by LaClaire et al. revealed that not only was there incorrect serotype identification at state health departments but also that 68% of the isolates recognized by slide agglutination as Hib did not have capsular genes when identified by PCR. The authors concluded, in accordance with our observation, that the real number of Hib cases, including vaccine failures, may be considerably lower than actually notified (16).

In our study resistance to ampicillin was exclusively connected with serotype b and with the production of ß-lactamase only. The percentage found in our study (14.6%) is similar to the results of the Japanese study, in which 15.4% of the meningitis isolates produced ß-lactamase but, exceptionally, 44.5% of the isolates were resistant to ampicillin due to changes in penicillin-binding proteins (PBPs; ß-lactamase-nonproducing ampicillin-resistant isolates [BLNAR]). What is more worrying is that 10.9% of H. influenzae isolates were resistant as a result of both mechanisms, having altered PBPs and producing ß-lactamase (11). Although we did not find any meningitis BLNAR isolates, the prevalence of such isolates, although rare until recently, is increasing among those responsible for lower respiratory tract infections in Poland, as well as in some other European countries (8). Many countries, such as the United States before the vaccination era and presently Cuba, Bangladesh, and Paraguay, reported a high percentage of ß-lactamase-producing isolates: 32%, 46.5%, 32.5%, and 30%, respectively (2, 26, 31, 34). In the light of the above data, the percentage of ß-lactamase producers among Polish meningitis isolates is not very high, although it is three times higher than found among H. influenzae isolates responsible for lower respiratory tract infections in Poland (28). The observation that antibiotic resistance is more prevalent in encapsulated isolates, responsible for the majority of meningitis cases, than in nontypeable H. influenzae was also revealed by other authors (4, 10, 32). The results of some studies showed that there is also an increasing number of H. influenzae isolates resistant to chloramphenicol (e.g., Bangladesh, 21.5%; Paraguay, 20%; Cuba, 44%) and an increasing number of isolates resistant simultaneously to both mentioned antibiotics (2, 26, 31). Although in our study nonsusceptibility to chloramphenicol was present in less than 2% of the isolates, all of them were simultaneously resistant to ampicillin.

In the present study all Hib isolates showed a high genetic homogeneity, because even the identification of 5 different PFGE types did not exclude the possible relatedness of all the Hib isolates tested. Additionally, the most common PFGE type, A, characterized more than 95% of the Hib isolates, and the most common PFGE subtype, A1, was also the most prevalent among isolates responsible for invasive disease in Italy and the Czech Republic and among isolates causing lower respiratory tract infections in Poland (28, 32). Thus, the results of this study support the evidence that the genetic structure of encapsulated H. influenzae is clonal. This fact is especially well known for Hib isolates, which are genetically homogenous and which the relationship has been found even for isolates originating from different countries and from different decades (18, 21, 32). However, in The Netherlands an increase in the genetic diversity of the Hib population after the introduction of vaccination against Hib has been reported recently (27).

The PFGE method is still widely used in bacterial typing. However, its usage is limited because of the difficulty in comparing results from different laboratories. Such comparisons can be made by MLST, i.e., by sequencing an internal fragment of usually seven housekeeping genes (www.mlst.net) (6, 17). An MLST scheme has been developed recently for H. influenzae isolates (www.mlst.net) (18). This technique, used in the present study, revealed six STs previously described and four new STs among the Polish encapsulated H. influenzae isolates tested. The most prevalent ST among the Hib isolates was ST6, which had been previously observed in the United States, Norway, Russia, The Netherlands, and the Czech Republic (www.mlst.net) (15, 18, 27). In the last country, 22 out of the 26 Hib isolates tested belonged to the ST6 (15). In the Dutch study, in which 241 Hib isolates were analyzed by MLST, 76.3% of them belonged to ST6 (27). The second most common ST in our study, ST92, an SLV of ST6 in the recA locus, was found in Russia, whereas ST44 was found in the United States, England, Ghana, Kenya, and The Netherlands (www.mlst.net) (18, 27). Other STs present in our study, ST80, -93, and -95, were found in Russia previously, and, additionally, ST80 was found in Kyrgyzstan. The new ST, ST190, represented in our study by two isolates, was described by Schouls et al. as the only ST absent in a prevaccination collection of isolates but present in 12 isolates from the postvaccination era. Additionally, four of these isolates were responsible for vaccine failures (27).

The present study is the first analysis of Polish Hib meningitis isolates using molecular techniques, including MLST, and thereby allows the inclusion of Polish data in the international database to track the spread of invasive and resistant clones of Hib. It also contributes to an understanding of the global epidemiology of Hib; although its importance is very limited in the countries with mass vaccination, it is important in others where Hib is still one of the most common bacterial etiologic agents of invasive infections in children. The presented data underline the urgent need for mass vaccination against Hib in Poland, extending beyond the risk groups.

 
We are grateful to Paula Kriz for the provision of the Czech H. influenzae serotype b isolate and to Marina Cerquetti for the Italian isolate. We thank all clinicians and microbiologists for providing isolates and clinical data for the NRCBM. We thank Anna Klarowicz for her excellent technical assistance and Cara Horowitz for English language editing.

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

 
* Corresponding author. Mailing address: National Reference Centre for Bacterial Meningitis, Dept. of Epidemiology and Clinical Microbiology, National Institute of Public Health, Chelmska 30/34, 00-725 Warsaw, Poland. Phone: 48 22 8514670. Fax: 48 22 8412949. E-mail: skoczek{at}cls.edu.pl.

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Journal of Clinical Microbiology, November 2005, p. 5665-5669, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5665-5669.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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