European Journal of Clinical Microbiology & Infectious Diseases

, Volume 33, Issue 4, pp 603–610

Characteristics of Haemophilus influenzae invasive isolates from Portugal following routine childhood vaccination against H. influenzae serotype b (2002–2010)

Authors

    • National Reference Laboratory for Bacterial Respiratory Infections, Department of Infectious DiseaseNational Institute of Health Dr. Ricardo Jorge
  • A. S. Simões
    • Laboratory of Molecular Microbiology of Human PathogensInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa
  • C. R. Betencourt
    • National Reference Laboratory for Bacterial Respiratory Infections, Department of Infectious DiseaseNational Institute of Health Dr. Ricardo Jorge
  • R. Sá-Leão
    • Laboratory of Molecular Microbiology of Human PathogensInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa
  • The Portuguese Group for the Study of Haemophilus influenzae invasive infection
Article

DOI: 10.1007/s10096-013-1994-6

Cite this article as:
Bajanca-Lavado, M.P., Simões, A.S., Betencourt, C.R. et al. Eur J Clin Microbiol Infect Dis (2014) 33: 603. doi:10.1007/s10096-013-1994-6

Abstract

We aimed to characterize Haemophilus influenzae invasive isolates recovered in Portugal over a 9-year period (2002–2010) following the inclusion of H. influenzae serotype b (Hib) conjugate vaccination in the National Immunization Program (NIP) in the year 2000 and compare the results with those obtained in a similar study from the pre-vaccination era (1989–2001) previously described by us. As part of a laboratory-based passive surveillance system, 144 invasive isolates obtained in 28 Portuguese hospitals were received at the National Reference Laboratory for Bacterial Respiratory Infections and were characterized. Capsular types and antibiotic susceptibility patterns were determined. The ftsI gene encoding PBP3 was sequenced for β-lactamase-negative ampicillin-resistant (BLNAR) isolates. Genetic relatedness among isolates was examined by multilocus sequencing typing (MLST). Most isolates (77.1 %) were non-capsulated, a significant increase compared to the pre-vaccination era (19.0 %, p < 0.001). Serotype b strains decreased significantly (from 81.0 to 13.2 %, p < 0.001) and serotype f increased significantly (from 0.8 to 6.9 %, p = 0.03). Ten percent of the isolates were β-lactamase producers, a value lower than that previously observed (26.9 %, p = 0.005). Eight percent of all isolates were BLNAR. A high genetic diversity among non-capsulated isolates was found. By contrast, capsulated isolates were clonal. The implementation of Hib vaccination has resulted in a significant decline in the proportion of serotype b H. influenzae invasive disease isolates. Most episodes of invasive disease occurring in Portugal are now due to fully susceptible, highly diverse, non-capsulated strains. Given the evolving dynamics of this pathogen and the increase in non-type b capsulated isolates, continuous surveillance is needed.

Introduction

Haemophilus influenzae can cause life-threatening infections in children and adults, such as pneumonia, bacteremia, and meningitis [1, 2]. Six capsular types, a–f, have been identified to date. Non-capsulated H. influenzae (NCHi) have also been described. H. influenzae serotype b (Hib) has long been a major cause of morbidity and mortality, especially in children less than 5 years of age [1]. However, the introduction of Hib conjugate vaccination has led to the near extinction of Hib disease [1]. This, in turn, has resulted in an increase in non-Hib disease, especially of NCHi in all age groups [1, 2]. In Portugal, Hib vaccination was implemented in the National Immunization Program (NIP) in June 2000 for all children of pre-school age (≤5 years old). The vaccine schedule is three doses at 2, 4, and 6 months of age, and a booster dose at 18 months.

β-lactams have long been used to treat H. influenzae infections, but resistance has emerged and disseminated. Two β-lactam resistance mechanisms have been described: one involves enzymatic hydrolysis of β-lactams by TEM-1 or ROB-1 β-lactamases [3]. The other involves decreased β-lactam affinity for penicillin binding protein 3 (PBP3) due to alteration in the ftsI gene [4]. The gold standard methodology to characterize strains exhibiting this non-enzymatic mechanism of resistance is sequencing of the ftsI gene. However, this can be challenging, as some ampicillin-susceptible strains already have mutations in ftsI [46]. Phenotypically, H. influenzae have been designated according to their resistance mechanism and Clinical and Laboratory Standards Institute (CLSI) breakpoints [7], such as β-lactamase non-producing ampicillin-susceptible (BLNAS), β-lactamase non-producing ampicillin-resistant (BLNAR), and β-lactamase producing ampicillin-resistant (BLPAR) strains [8].

An accurate molecular characterization of H. influenzae invasive disease isolates is important to monitor and understand changes in the epidemiology of these bacteria, particularly following a massive intervention, such as the introduction of Hib conjugate vaccination. Multilocus sequencing typing (MLST), a molecular typing method based on the sequence of internal fragments of seven housekeeping genes, has been described and validated for H. influenzae and has been increasingly used [9].

The aim of this work was to characterize H. influenzae invasive isolates in Portugal over a 9-year period (2002–2010) following the introduction of Hib conjugate vaccination. As a secondary objective, we aimed to compare the findings with results from the pre-vaccination era (1989–2001) previously reported [10].

Materials and methods

Bacterial isolates

The National Reference Laboratory for Bacterial Respiratory Infections (NRLBRI), based at the National Institute of Health in Lisbon, is the reference laboratory for H. influenzae, being responsible for the surveillance of invasive H. influenzae infections. A laboratory surveillance system was established on a voluntary basis in 1989. Throughout the study, 28 hospitals, from all regions of the country, isolated and identified H. influenzae invasive isolates, and sent them to the NRLBRI to be further characterized.

Between January 1, 2002 and December 31, 2010, 144 H. influenzae invasive isolates were received. The isolates originated from 12 hospitals from the north, ten from the center, four from the south, and two from the islands. The clinical sources of the isolates were cerebrospinal fluid (CSF) (n = 29), blood (n = 109), and pleural fluid (n = 6). Males accounted for 59 % of the cases. Forty-one isolates were from pre-school children, 15 from older children (6–18 years old), and 86 were from adults (>18 years old).

Upon arrival, all strains were cultured on chocolate agar supplemented with polyvitex (bioMérieux, Linda-a-Velha, Portugal) and incubated at 35 °C for 18–24 h in 5 % CO2 atmosphere. Pure cultures were stored in tryptic soy broth with 20 % glycerol at −80 °C.

Preparation of DNA templates for PCR amplification

Six to eight colonies of an overnight culture were resuspended in 60 μl of sterile water and boiled for 15 min. After centrifugation at 12,000g, the supernatant was removed and used as template.

Capsular typing

The capsular status was identified by polymerase chain reaction (PCR) amplification of the bexA gene (responsible for capsule transport) and the capsular type was determined by amplification of capsule-specific genes (for serotypes a–f) using primers and conditions described previously [11].

Testing of antimicrobial susceptibility and β-lactamase production

The minimum inhibitory concentration (MIC) for ampicillin, amoxicillin/clavulanic acid, cefaclor, cefuroxime, cefotaxime, cefepime, meropenem, ciprofloxacin, azithromycin, chloramphenicol, tetracycline, rifampin, and trimethoprim–sulfamethoxazole were determined by the broth microdilution method (Siemens Healthcare Diagnostics Lda, Amadora, Portugal) in Haemophilus test medium (HTM) according to the CLSI guidelines [7]. β-lactamase production was screened by the chromogenic cephalosporin assay using nitrocefin as subtract (Oxoid Limited, Hampshire, UK). Strains ATCC49247 (BLNAR), ATCC10211 (BLNAS), and NCTC11315 (BLPAR) were used as controls.

Screening for ampicillin-resistant determinants

The presence of the β-lactamase encoding blaTEM-1 gene was investigated in all β-lactamase producing isolates by PCR using primers and conditions previously described [12]. Alterations in PBP3 were investigated in β-lactamase negative isolates with an MIC to ampicillin ≥1 mg/L by sequencing the region of the transpeptidase domain of ftsI as previously described [13]. Sequences were compared with those of H. influenzae RD KW20 (accession number NC_000907).

MLST

MLST was performed as previously described [9]. Sequences were analyzed and submitted to the MLST website (http://haemophilus.mlst.net) for the assignment of sequence type (ST). goeBURST analysis was performed using PHYLOVIZ 1.0 (http://www.phyloviz.net).

Results

Capsular serotypes

One hundred and eleven of 144 isolates (77.1 %) were NCHi. Capsulated strains were of serotypes b (19/144, 13.2 %), f (10/144, 6.9 %), a (3/144, 2.1 %), and d (1/144, 0.7 %). Table 1 shows the distribution of serotypes per year.
Table 1

Distribution of H. influenzae serotypes by year

Year

No. of isolates

Serotype (no. of isolates)

2002

11

NCHi (11)

2003

12

b (3), NCHi (9)

2004

19

a (2), b (6), f (1), NCHi (10)

2005

14

b (2), f (2), NCHi (10)

2006

17

a (1), f (1), NCHi (15)

2007

11

b (1), NCHi (10)

2008

12

b (1), f (2), NCHi (9)

2009

20

b (2), d (1), f (2), NCHi (15)

2010

28

b (4), f (2), NCHi (22)

NCHi strains were isolated from children (n = 38, 29 from blood, six from CSF, and three from pleural fluid) and adults (n = 72, 59 from blood, 11 from CSF, and two from pleural fluid). For one NCHi isolated from pleural fluid, the patient’s age was unavailable. Strains of serotype a were isolated from children (two from CSF and one from blood). Strains of serotype b were isolated from children (seven from CSF and four from blood) and adults (one from CSF and seven from blood). Serotype f strains were isolated from children and adults (four each, all were isolated from blood). Among the children who suffered from Hib invasive disease, seven were aged up to 2 years old, two were older, and for two, the age was unknown. The vaccination status was only known for six children, all aged less than 2 years old: five were vaccinated with Hib vaccine and one was not vaccinated. There was no information on underlying conditions.

Antimicrobial susceptibility testing and β-lactamase production

Antimicrobial susceptibility testing was performed for 142 isolates (Table 2). Ampicillin resistance (intermediate and full resistance) was found in 14.1 % (n = 20) of isolates (15 were BLPAR, five were BLNAR). Non-susceptibility to cefuroxime, cefaclor, and tetracycline was detected in 2.1, 7.0, and 2.1 % of the isolates, respectively. For trimethoprim–sulfamethoxazole, 20.4 % of the isolates were non-susceptible. All isolates were susceptible to amoxicillin/clavulanic acid, cefotaxime, cefepime, meropenem, ciprofloxacin, azithromycin, chloramphenicol, and rifampin.
Table 2

Antimicrobial susceptibility of invasive H. influenzae isolates

Antimicrobial agent

MIC (mg/L)

Susceptibility class (%)

MIC50

MIC90

MIC range

S

I

R

Ampicillin

0.25

8

≤0.03–64

85.9

3.5

10.6

Amoxicillin/clavulanic acid

0.5

1

≤0.25–4

100

0

0

Cefepime

≤0.12

0.25

≤0.12–2

100

0

0

Cefotaxime

≤0.03

0.06

≤0.03–0.5

100

0

0

Cefuroxime

0.5

2

≤0.25 to >8

97.9

1.4

0.7

Cefaclor

2

8

≤1 to >16

93

4.2

2.8

Meropenem

≤0.06

0.12

≤0.06–0.25

100

0

0

Ciprofloxacin

≤0.06

0.12

≤0.06–0.25

100

0

0

Azithromycin

1

2

0.25–2

100

0

0

Chloramphenicol

≤1

≤1

≤1

100

0

0

Tetracycline

≤1

≤1

≤1 to >4

97.9

0.7

1.4

Rifampin

≤0.5

≤0.5

≤0.5–1

100

0

0

Trimethoprim–sulfamethoxazole

≤0.25

>2

≤0.25 to >2

79.6

2.1

18.3

Resistance profiles within the H. influenzae serotypes are shown in Table 3. Resistance was associated to NCHi isolates (41/109, 37.6 %) when compared with serotypeable isolates (3/33; 9.1 %).
Table 3

Antimicrobial resistance profile within H. influenzae serotypes

Serotype

No. of isolates

Antimicrobial resistant pattern

a

3

Susceptible to all

b

1

Amp, Tet (I)

 

2

SXT

 

16

Susceptible to all

d

1

Susceptible to all

f

10

Susceptible to all

NCHi

6

Amp

 

5

Amp, SXT (I + R)

 

2

Amp, Cef (I)

 

1

Amp, Cfx (I), Cfc (I)

 

2

Amp (I)

 

2

Amp (I), SXT

 

1

Amp (I), Cfx (I), Cef (I), SXT

 

17

SXT (I + R)

 

1

SXT, Tet

 

1

Cfx (I), Cef (I), SXT

 

1

Tet

 

2

Cef (I)

 

68

Susceptible to all

I, intermediate; R, resistant; Amp, ampicillin; Tet, tetracycline; SXT, trimethoprim–sulfamethoxazole; Cef, cefaclor, Cfx, cefuroxime

Genetic determinants of β-lactam resistance

Screening of the 15 BLPAR isolates for the presence of TEM showed that all strains harbored a blaTEM-1 gene.

For the characterization of the five BLNAR isolates, the transpeptidase domain of the ftsI gene was analyzed. In addition, there were seven isolates that had MICs to ampicillin of 1 mg/L that were also analyzed, as mutations in the ftsI gene have been associated with these MICs [5, 14]. Eleven isolates had one or more non-synonymous mutations in the transpeptidase domain, being classified as gBLNAR (Table 4). One isolate (ampicillin MIC = 2 mg/L) had no mutations in ftsI. The most frequent amino acid substitutions were Asn526Lys (10/11, near the KTG motif) and Asp350Asn (9/11) [5, 15].
Table 4

Amino acid substitutions identified in the transpeptidase domain of the ftsI gene of gBLNAR isolates

Groupa

No. of β-lactamase non-producing isolatesb

Amino acid substitution

 

Near SSN motif

   

Near KTG motif

  

Asp350

Met377

Ala437

Ile449

Gly490

Ala502

Arg517

Asn526

Ala530

Thr532

Val547

Asn569

I

1

Asn

Ile

    

His

  

Ser

Ile

 

IIb

1

Asn

 

Ser

Val

   

Lys

  

Ile

Ser

 

2

Asn

Ile

   

Val

 

Lys

  

Ile

Ser

1

Asn

Ile

  

Glu

Val

 

Lys

  

Ile

Ser

3

Asn

   

Glu

  

Lys

Ser

   

1

     

Val

 

Lys

    

1

Asn

      

Lys

  

Ile

Ser

1

    

Glu

Val

 

Lys

  

Ile

Ser

aGroups and subgroups were defined according to Ubukata et al. [5] and Dabernat et al. [15]

bOne of the study strains (8760, not shown) had no substitutions in the ftsI gene, despite having an MIC of 2 mg/L to ampicillin and amoxicillin/clavulanic acid

Molecular genotyping by MLST

One hundred isolates were typed by MLST. The following selection criteria were used: (i) include all capsulated isolates (n = 33); (ii) include representatives of all resistance patterns detected among NCHi isolates. In this latter group, when a resistance pattern was associated with up to ten isolates, all of them were selected. For resistance patterns with >10 isolates, at least five were selected. Within each resistance pattern, strains were chosen to cover the diversity of years and hospitals.

Overall, 13 novel alleles were identified (atpG, 110–112, 119; fucK, 95; mdh, 227; pgi, 210, 211, 213, 218; and recA, 131–133) and 29 novel STs (1135–1154, 1159, 1160, 1188, 1189, and 1227–1231) were assigned. In total, 71 STs were identified and these were distributed into nine clonal complexes (CC) and 42 singletons (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10096-013-1994-6/MediaObjects/10096_2013_1994_Fig1_HTML.gif
Fig. 1

goeBURST representation of H. influenzae invasive disease isolates. The numbers indicate sequence types; lines between sequence types connect single-locus variants. The size of the sequence type is proportional to the number of isolates in each sequence type. Associated serotypes are indicated above the sequence type/clonal complex; the remaining ones are non-capsulated. The asterisks denote new sequence types described in this study. A dashed circle around a sequence type indicates gBLNAR isolates, while a solid circle indicates BLPAR isolates, and a square indicates a BLNAR isolate. For sequence type 103, one of the four isolates was not BLPAR

Capsulated isolates of serotypes b (n = 19) and f (n = 10) were highly clonal: among serotype b, all isolates but one belonged to CC6, and all isolates of serotype f belonged to CC124. In contrast, NCHi isolates were very diverse: 42 out of 67 isolates were singletons. Still, six clonal complexes were found: CC3 (n = 4), CC57 (n = 4), CC103 (n = 7), CC107 (n = 5), CC165 (n = 3), and CC244 (n = 2) (Fig. 1). Each ST and CC was only associated with a single capsular type.

All but one of the BLPAR isolates (n = 15) were NCHi and were distributed across four CCs. The other isolate was a serotype b associated with a singleton. The gBLNAR (n = 11) isolates were all NCHi and were associated with several STs (Fig. 1).

Discussion

Studies on H. influenzae invasive disease in Portugal are very scarce and temporal trends have been described only once for the period 1989–2001 [10]. In that study, the authors described changes in capsular type and antimicrobial resistance among invasive disease isolates obtained in three periods encompassing the pre-vaccination era (1989–1993), the period when Hib vaccination was available but not part of the NIP (1994–1999), and the first 18 months following its introduction into the NIP (2000–2001). In the current study, we aimed to describe the characteristics of H. influenzae isolates causing invasive disease in Portugal in the 9-year period following implementation of the vaccination in the NIP (2002–2010).

We observed that, in 2002–2010, Hib strains accounted for 13.2 % of the invasive disease, a value which is significantly lower than that observed in the pre-vaccination era (81.0 %, p < 0.001) and comparable to the first 18 months following the incorporation of Hib vaccination into the NIP (16.0 %, p = 0.71) [10]. In addition, in 2002–2010, most cases (77.1 %) of H. influenzae invasive disease were caused by NCHi isolates, a significant increase compared to the pre-vaccination era (19.0 %, p < 0.001), and comparable to the first 18 months following inclusion of the Hib vaccine in the NIP (80.0 %, p = 0.75) [10]. Elsewhere, the widespread adoption of this vaccine has also resulted in a significant decline of Hib disease in children and a concomitant shift towards NCHi invasive disease [1622].

Based on the analysis of our collection, the data suggest the emergence of non-b capsular types. While in the pre-vaccine period non-b capsular types were not detected, and the first non-b capsulated invasive isolate was detected only in 2001 (serotype f) [10], in the current study, isolates of serotypes f (n = 10), a (n = 3), and d (n = 1) were identified. Serotype f has also been described as a cause of invasive disease in other European countries [2325]. The case due to serotype d was the first case of meningitis in an infant in Europe due to such type, and has been recently described [26]. Although rarely, this serotype has been described in Europe, in older children and adults [27], and it has been generally regarded as a serotype not associated to disease.

Antibiotic resistance has been an issue of concern, especially with the increasing description of BLNAR strains worldwide [2835]. The detection of this phenotype is clinically relevant, as these strains have lower responsiveness to β-lactams and are not targeted by clavulanic acid [8, 36]. In Portugal, this phenotype has been described at least since 1992 and has been mostly associated to respiratory infections [37, 38]. In this study, we identified 11 gBLNAR strains (ampicillin MIC of 1–2 mg/L), all with relevant mutations in ftsI [5, 15]. Although strains with ampicillin MIC = 1 mg/L are not considered resistant by current CLSI breakpoints [7] nor European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [39, 40], our findings are in agreement with other studies which have detected mutations in the ftsI gene of these strains, resulting in PBP3 substitutions responsible for the decreased susceptibility to ampicillin [5, 14, 15, 36, 41, 42]. In fact, the CLSI and EUCAST classification of ampicillin resistance of H. influenzae isolates differ only for an MIC of 2 mg/L, which is classified by the former in the intermediate category and by the latter in the resistance category [7, 39, 40].

In 2007, a Spanish study described gBLNAR isolates with III-like pattern mutation in the ftsI gene that had MICs to ampicillin lower than 1 mg/L but increased MICs to cephalosporins [43]. However, a screening of over 500 isolates of H. influenzae (including susceptible, intermediate, and resistant isolates to ampicillin) did not reveal isolates with such characteristics in Portugal [14; Lavado, unpublished]. In the current study, we identified three isolates with such phenotype (increased MICs to cephalosporins but ampicillin MIC lower than 1 mg/L), but based on our previous observations, the ftsI gene has not been sequenced for these isolates.

We have observed that 10.4 % of the isolates were β-lactamase producers, a value significantly lower than that observed in the previous study (26.9 %, p = 0.005) [10]. In both studies, β-lactamase production was confined to NCHi and type b isolates. Although the prevalence of these types changed significantly between studies, this could not account for the differences in resistance obtained: in the previous study, β-lactamase production was observed in 31.9 % and 19.6 % of the type b and NCHi strains, respectively; the corresponding numbers in the current study were 5.3 % and 12.6 %, respectively. Although high rates of β-lactamase producing strains have been recently described in countries like Japan [44], a decreasing prevalence of strains with this resistance mechanism has also been noticed in Europe and the United States [28, 33, 45].

Regarding other antibiotics, a pronounced decline in non-susceptibility to tetracycline (16.0 % vs. 2.1 %) and chloramphenicol (10.1 % vs. 0 %) was noted. This could be attributed to the decline in serotype b strains, as they were previously associated with this resistance phenotype. In addition, in the current study, there were no isolates resistant to third-generation cephalosporins, the first choice in the treatment of H. influenzae disease.

Genotyping by MLST revealed a very high diversity [Simpson’s Diversity Index of 0.977; 95 % confidence interval (CI) 0.963-0.992] among non-capsulated isolates translated in 42 singletons and six CCs. By contrast, capsulated isolates were very clonal. For instance, all but one isolates of serotype b clustered in CC6. These observations are in line with other studies that have also noticed the disparity in diversity between NCHi and capsulated H. influenzae [9, 10]. In fact, among the NCHi isolates, no dominant clone was observed; the most abundant CC accounted for only 6.3 % of the NCHi isolates. Furthermore, no correlation between ST/CC and the pattern of antimicrobial resistance was found among NCHi isolates. The same evidence has been described by Resman et al. [29] and has been justified by the observation that NCHi are highly recombinogenic, suggesting that the acquisition of resistance can occur independently multiple times [46].

Of interest, although 40.8 % of the 71 STs were newly described, the remaining STs, accounting for 71 % of the isolates, have been described in other countries, suggesting a common ancestor between them.

No capsular switching events were observed in our collection, i.e., there was no ST/CC associated to more than one serotype. Also, all previously described STs had the same serotype association previously reported.

Our study has one important limitation. Due to the nature of the laboratory-based surveillance system in place, we have characterized a convenience sample and were unable to calculate the incidence of invasive H. influenzae disease. Still, we have received isolates from hospitals from all over the country and, therefore, our results should be representative of the properties of the population of H. influenzae causing invasive disease. In addition, as there have been no other studies from Portugal reporting the characteristics associated with H. influenzae causing invasive disease during the last decade, our results are novel and relevant for surveillance.

In conclusion, we have shown that the implementation of Hib vaccine in the NIP has resulted in a marked reduction on the proportion of Hib isolates causing invasive disease. A marked reduction in antibiotic resistance, including ampicillin resistance, has also been observed. Most cases of H. influenzae invasive disease occurring in Portugal are now due to fully susceptible NCHi strains. In the years to come, it will be important to continue monitoring this disease. Finally, efforts to implement a better surveillance program should be made.

Acknowledgments

Members of the Portuguese Group for the Study of Haemophilus influenzae invasive infection are: Ana Maria de Jesus (Centro Hospitalar do Barreiro Montijo), Henrique Oliveira, Luís Albuquerque, Sarabando Moreira (Centro Hospitalar de Coimbra), Rosa Barros, Isabel Peres, João Marques (Centro Hospitalar de Lisboa Central), José Melo-Cristino, Luís Lito, Lurdes Monteiro, Ana Fonseca, José Gonçalo Marques (Centro Hospitalar Lisboa Norte), Filomena Martins (Centro Hospitalar de Lisboa Ocidental), Fernanda Teixeira, (Centro Hospitalar do Porto), Margarida Tavares, Artur Bonito Vítor (Centro Hospitalar de S. João), Cláudia Monteiro, Mariana B. Viana (Centro Hospitalar Tâmega e Sousa), Paulo Lopes, Luísa Felício (Centro Hospitalar Vila Nova de Gaia-Espinho), Maria João Virtuoso (Hospital de Faro), Amélia Cavaco, Teresa Afonso (Hospital Dr Nélio Mendonça, Funchal), Adriana Coutinho (Hospital Espírito Santo, Évora), José Diogo (Hospital Garcia da Orta, Almada), Maria Manuel Flores, Paula Reis, Elmano Ramalheira (Hospital Infante D. Pedro, Aveiro), Luísa Sancho, Teresa Sardinha, Paula Correia (Hospital Professor Doutor Fernando da Fonseca, Amadora), Sandra Vieira (Hospital de Santa Luzia de Viana do Castelo), José Miguel Ribeiro (Hospital de São Teotónio, Viseu), Maria Augusta Santos (Instituto Nacional de Saúde, Porto), Florbela Cunha, Margarida Rodrigues (Hospital Reynaldo dos Santos, Vila Franca de Xira), Luísa Cabral (Hospital dos SAMS, Lisboa), Ana Paula Castro (Hospital de Vila Real).

We thank Manuela Caniça, Head of the National Reference Laboratory for Antimicrobial Resistance, from the National Institute of Health, Lisbon, Portugal, for providing us with some Haemophilus influenzae strains.

Funding

This work was supported by the National Institute of Health, Lisbon, Portugal.

Conflict of interest

The authors declare that they have no conflict of interest.

Copyright information

© Springer-Verlag Berlin Heidelberg 2013