Antibiotic resistance and clinical significance of Haemophilus influenzae type f

José Campos1,*, Federico Román1, María Pérez-Vázquez1, Belén Aracil1, Jesús Oteo1, Emilia Cercenado2 and the Spanish Study Group for H. influenzae type f§

1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ministry of Health, 28220 Majadahonda, Madrid; 2 Servicio de Microbiología, Hospital Gregorio Marañón, Madrid, Spain

Received 4 July 2003; returned 27 September 2003; revised 30 September 2003; accepted 3 October 2003


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives: Little is known about the antibiotic susceptibility and clinical significance of non-type b capsulated Haemophilus influenzae. We studied the antibiotic resistance patterns, plasmid carriage and clinical features of H. influenzae type f infections in Spain during 1996–2002.

Patients and methods: Forty-nine H. influenzae type f recovered from Spanish hospitals were analysed at a central laboratory where full microbiological and molecular epidemiological studies were carried out. Antimicrobial susceptibility testing was performed in accordance with NCCLS guidelines.

Results: Twelve strains (24.5%) were resistant to ampicillin and 22 (44.9%) to co-trimoxazole. Decreased susceptibility to clarithromycin, tetracycline, chloramphenicol and rifampicin was found in 16.3%, 12.2%, 14.3% and 2% of strains, respectively. Multidrug resistance was present in nine (18.4%) of the 49 isolates. The most prevalent resistance phenotype was ampicillin/tetracycline/co-trimoxazole/chloramphenicol, which was detected in five isolates. All six strains that were simultaneously resistant to ampicillin, tetracycline and chloramphenicol had conjugative plasmids. The main clinical diagnoses were pneumonia (32.6%), sepsis (18.4%) and meningitis (16.3%). Thirty-two patients (65.3%) had previous underlying predisposing conditions, principally respiratory diseases (20.4%). Twenty-one patients (42.8%) had impaired immunity. Thirty-seven (75.5%) patients were >14 years old, 12 (24.5%) were <=14 years, and seven were <=5 years. Most isolates were clonally related.

Conclusions: A high prevalence of antibiotic resistance, including multiresistance, was detected in Spanish H. influenzae type f isolates. Carriage of large conjugative plasmids was strongly associated with antibiotic resistance. H. influenzae type f is mainly an opportunistic pathogen, although it may cause primary severe infections, such as meningitis in children.

Keywords: H. influenzae, plasmid carriage, opportunistic infections


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Haemophilus influenzae is a frequent inhabitant of the human upper respiratory tract and a strictly human pathogen. It has been recognized as an important cause of a variety of severe clinical conditions such as pneumonia, bacteraemia, meningitis, epiglottitis, septic arthritis and cellulitis.1

H. influenzae can be classified into six capsular types (a–f) and non-typeable strains according to its capsular antigen composition. H. influenzae type b is the most invasive capsular type and was once one of the most prevalent bacterial pathogens, causing meningoencephalitis and other invasive infections in children below the age of 5 years. Since the early 1990s, the widespread use of H. influenzae type b conjugate vaccines in western countries has had a dramatic effect on preventing invasive infections due to this organism.2,3

However, such widespread vaccination has raised concerns about the possibility of serotype replacement, as other serotypes and/or non-typeable strains may fill the ecological niche left open by H. influenzae type b.4 It has been suggested that H. influenzae type f could be a new emerging pathogen.5 Serotype information is essential to evaluate vaccine efficacy and eventual changes in the epidemiology of H. influenzae infections.2 Recently, the CDC have recognized important serotype discrepancies between standard slide agglutination typing and PCR-based capsule typing.6,7 Some authors have noted a trend towards an increased incidence of H. influenzae types e and f after widespread vaccination against H. influenzae type b.8,9

Little information is available concerning the clinical, microbiological and epidemiological significance of H. influenzae type f. Most published reports are of individual clinical cases of meningitis, septic arthritis, osteomyelitis, pneumonia and sepsis.921

During 1996–2002, we studied the antibiotic resistance patterns, plasmid carriage and clinical features of H. influenzae type f infections in Spain. Additionally, we investigated whether the IS1016- bex A deletion (a virulence factor found in invasive H. influenzae type b) had been acquired by H. influenzae type f.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In 1996, vaccination against H. influenzae type b with conjugate vaccine was introduced into the regular vaccination programme, first in a few areas of Spain and then nationally. As part of a Europe-wide study, the Spanish national Haemophilus reference laboratory set up an active programme to characterize the phenotype and genotype of H. influenzae type b and other capsular types. Clinical microbiology laboratories, representing all geographical areas, aimed to send recovered H. influenzae to the reference laboratory. H. influenzae type f strains were analysed at our central laboratory, where full microbiological identification, susceptibility testing and molecular epidemiological studies were carried out. When an isolate was identified as H. influenzae type f, the clinical records of the patient were examined retrospectively, and a further clinical record was completed, which included the patient’s identification data, diagnosis, underlying conditions and outcome.

Identification was confirmed according to standard laboratory methods.1 These included Gram stain, X and V factors, ornithine, urea, indole and porphyrin tests. Two methods were used to carry out capsular typing in all isolates: the classic slide agglutination test, using type-specific antiserum (Difco Laboratories, Detroit, MI, USA), and the PCR method of Falla et al.,22 which was considered the reference test.

Antimicrobial susceptibility testing was performed in accordance with the guidelines of the NCCLS,23 using a Haemophilus test medium with a semi-automated commercialized microdilution method (Wider; Fco. Soria Melguizo S.A., Madrid, Spain). The antimicrobial agents tested were ampicillin, co-amoxiclav, cefuroxime, cefotaxime, cefixime, cefepime, meropenem, clarithromycin, tetracycline, ciprofloxacin, levofloxacin, co-trimoxazole, chloramphenicol and rifampicin. H. influenzae ATCC 49247 and H. influenzae ATCC 49766 were used as quality control strains.

ß-Lactamase production was determined by the chromogenic cephalosporin test with nitrocephin as substrate. Chloramphenicol acetyl transferase was measured as described previously.24

To determine whether a putative virulence mutation genotype, described in H. influenzae types b and a,25 had been acquired by H. influenzae type f, the IS1016-bex A deletion was amplified by PCR, as previously described;25 the Eagan strain of H. influenzae type b was used as a positive control.

All 49 clinical strains of H. influenzae type f and the H. influenzae type f reference strain ATCC 9833 were examined by PFGE. This examination was conducted following digestion of bacterial DNA with SmaI (MBI Fermentas, Vilnius, Lithuania) and separation of the fragments in a 1% agarose gel prepared in 0.5 X TBE buffer with Bio-Rad CHEF Mapper apparatus (Bio-Rad, Madrid, Spain). Gels were stained with ethidium bromide and photographed under ultraviolet light. A genetic similarity dendrogram was designed and calculated from the Dice correlation coefficient, represented by UPGMA with Molecular Analysis Software (Bio-Rad) and with a tolerance level of 2%. Well-resolved bands, corresponding to fragments exceeding 48.5 kbp, were included in the computer analysis.

Screening of large conjugative plasmid carriage relating to antimicrobial resistance was carried out by PCR, as described by Leaves et al.26

The EPI-Info for Windows program, Release 6.04, was used for statistical analysis. Categorical variables were compared with Fisher’s exact test, two-tailed. The null hypothesis was rejected for values of P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
From January 1996–December 2002, a total of 6073 H. influenzae isolated from clinical samples were submitted to the national reference laboratory from all Spanish autonomous communities. Forty-nine (0.81%) of these strains were H. influenzae type f, both by molecular typing and agglutination. All belonged to biotype I, except two that were biotype II. The 49 isolates came from individual, unrelated patients from 18 institutions in seven geographical areas.

The MICs for the two ATCC control strains were always within recommended limits.23 Of the 49 isolates tested, 24.5% were resistant to ampicillin (all ß-lactamase producers) and 44.9% to co-trimoxazole (Table 1). No resistance to co-amoxiclav, cefuroxime, cefotaxime, cefixime, cefepime, meropenem, ciprofloxacin or levofloxacin was detected.


View this table:
[in this window]
[in a new window]
 
Table 1. Antimicrobial susceptibility of 49 H. influenzae type f isolates
 
A trend was found indicating that H. influenzae type f infections in children were more resistant than in adults to ampicillin (33.3% versus 21.6%) and co-trimoxazole (83.4% versus 59.4%), although the small number of cases prevents such differences being statistically significant.

Multidrug resistance (non-susceptibility to three or more antibiotics) was present in nine (18.4%) of the 49 isolates, and seven were isolated from patients who had at least one predisposing risk factor. The most prevalent resistance phenotype was ampicillin/tetracycline/co-trimoxazole/chloramphenicol, which was detected in five isolates, representing 55.5% of the multiresistant strains and 10.2% of strains overall. Of seven patients infected by isolates with simultaneous resistance to ampicillin and chloramphenicol, six were also resistant to tetracycline.

Overall carriage of large conjugative plasmids was detected by PCR in seven (14.3%) of the 49 H. influenzae type f strains. Its presence was strongly associated with antibiotic resistance, in particular with resistance to ampicillin, tetracycline and chloramphenicol. Plasmid was detected by PCR in all six strains that were simultaneously resistant to ampicillin, tetracycline and chloramphenicol. Seven of 12 ampicillin-resistant isolates (58.3%) had conjugative plasmids, whereas the 15 fully antibiotic susceptible strains did not (P < 0.001). Also, 100% of seven and six H. influenzae type f isolates resistant to chloramphenicol and tetracycline, respectively, carried conjugative plasmids (P < 0.001). In contrast, in the 21 isolates with resistance to co-trimoxazole and/or clarithromycin none carried them.

Twenty-four patients were male (49%) and 25 female (51%). Thirty-seven strains (75.5%) were isolated from patients aged >14 years, 12 (24.5%) from children <=14 years, seven of whom were 5 years old or younger (the age of five patients was unknown). The mean age was 43.3 years (range: 5 months–95 years; median: 42 years).

Twenty-five isolates (51%) were recovered from blood (18) or cerebrospinal fluid (7), 19 (38.8%) from respiratory tract samples (14 from sputum and five from bronchoalveolar aspirates), two (4.1%) from abscesses, and in one (2%) case each from conjunctiva, abdominal fluid and ear. The most frequent clinical diagnoses were pneumonia in 16 cases, sepsis in nine and meningitis in eight. Four of the 14 patients with H. influenzae type f in sputum were diagnosed with pneumonia, whereas the remaining 10 were diagnosed mainly with exacerbations of cystic fibrosis or chronic obstructive pulmonary disease. In some cases there was no clear evidence of any clinical implication and eventual colonization could not be excluded.

Seven (14.3%) patients died, all of whom were adults with severe underlying diseases. The antibiotic treatments most frequently used were third-generation cephalosporins and co-amoxiclav. Thirty-two patients (65.3%) had previous underlying predisposing conditions, and more than one risk factor was present in 13 of them. The most prevalent underlying diseases were respiratory diseases, in 10 cases, HIV infection in eight cases, tumoral pathologies in six cases, and liver pathology and diabetes in five cases each. Twenty-one (42.8%) patients had impaired immunity (HIV infection, Hodgkin’s lymphoma, multiple myeloma, IgG gammopathy, heart transplantation, leukaemia, corticosteroid treatments and diabetes).

Sixteen H. influenzae type f strains (32.6%) were isolated from a large hospital in Madrid with 1800 beds, covering a population of 640 000 inhabitants. In the community of Madrid (population more than 5 million), widespread vaccination with H. influenzae type b conjugate vaccines started in 1999. Over the study period, 2542 clinical isolates of H. influenzae were isolated in this institution, all of which were submitted to the reference laboratory, where 16 (0.6%) were identified as H. influenzae type f. In the area covered by this hospital, the overall incidence of H. influenzae infection during this period was 56.7 cases/100 000 inhabitants per year. In comparison, the incidence of H. influenzae type f infections was 0.40 cases/ 100 000 inhabitants per year. In the 7 full years included in the study, the incidence of H. influenzae type f infection was 0.31 cases/100 000 inhabitants in 1996, 0.62/100 000 in 1997, 0.16/100 000 in 1998, 0.47/100 000 in 1999, 0.31 cases/100 000 in 2000, 0.16/100 000 in 2001 and 0.47/100 000 in 2002. In the remaining hospitals with H. influenzae type f infections, the number of cases was too low to allow meaningful calculations.

As expected, the positive H. influenzae type b control (Eagan strain) presented the IS1016-bex A deletion when PCR-amplified. This was in contrast with the 49 H. influenzae type f strains, which did not present the deletion.

The genetic relatedness of 48 H. influenzae type f and the H. influenzae type f reference strain, estimated from the cluster analysis of the PFGE patterns obtained by DNA fingerprinting, is shown in Figure 1. The DNA of one isolate was not digested by SmaI. Overall, the Spanish H. influenzae type f population showed little genetic variability. Forty-four (91.6%) of the isolates had a genetic distance <=12% and could be considered to belong to the same clone according to Tenover criteria,27 irrespective of their geographical origin, antibiotic resistance patterns or clinical significance.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 1. Dendrogram illustrating the genetic relationship of 48 H. influenzae type f strains as determined by pulsed-field gel electrophoresis.

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Following the widespread use of effective conjugate vaccines, infections due to H. influenzae type b and the prevalence of H. influenzae type b carriers have decreased substantially.2,3 In Spain, widespread vaccination against H. influenzae type b has been in place since the mid-1990s. Vaccination campaigns create a novel epidemiological environment; it has been suggested that the decline in the rate of H. influenzae type b infections could have given rise to the emergence of diseases caused by other H. influenzae serotypes.8,9 An increased incidence of H. influenzae types e and particularly f has been noted in the USA.8,9

In Europe, little information is available about the antibiotic susceptibility patterns and clinical significance of H. influenzae type f. In general, information on H. influenzae type f infections is limited as only sporadic clinical cases of meningitis, septic arthritis, osteomyelitis, pneumonia and sepsis have been described in the literature.921 We have shown that H. influenzae type f may be the aetiological agent of a wide range of illnesses including mild infections (otitis, conjunctivitis) and severe infections (meningitis, pneumonia, sepsis).

Until now, very little information about antimicrobial susceptibility, other than to ampicillin, has been reported for H. influenzae type f. ß-Lactamase activity was detected in 21% of H. influenzae type f isolates studied by Urwin et al.8 In our study, 24.5% of 49 isolates produced ß-lactamase and resistance to other antibiotics like co-trimoxazole, chloramphenicol and tetracycline and multiple antibiotic resistance was also remarkable. Carriage of large conjugative plasmids was strongly correlated with the expression of multiple antibiotic resistance. The ampicillin resistance was similar to that described previously in studies on H. influenzae,2830 but clearly lower than the 50% and 61.5% found previously in Spain in H. influenzae types b and e, respectively.31,32

Carriage of high-molecular-weight conjugative plasmids in H. influenzae type b constitutes the genetic bases of resistance to ampicillin, chloramphenicol and tetracycline.33,34 In seven of our antibiotic-resistant H. influenzae type f isolates, conjugative plasmids were detected by PCR, and all six strains with cross-resistance to ampicillin, tetracycline and chloramphenicol had a conjugative plasmid, as revealed by PCR. Our data suggest that resistance to ampicillin, chloramphenicol and tetracycline, and multiple resistance, in H. influenzae type f is plasmid mediated, in contrast to co-trimoxazole and clarithromycin resistance.

In this study, we have shown that the epidemiology and clinical significance of H. influenzae type f infections is very different from the classic invasive infections caused by H. influenzae type b. Only a small proportion (14.3%) of cases occurred in children <=5 years, the majority being in patients of >14 years of age, often with predisposing factors and severe underlying conditions. In the series studied by Urwin et al.,8 26% of cases occurred in children younger than 5 years of age. We have found that the most frequent clinical presentations were infections of the respiratory tract (59.2%), these also being the commonest underlying diseases in infected patients (20.4%). However, meningitis was diagnosed mainly in adult patients (75%; 6/8), and in patients with predisposing factors (62.5%; 5/8).

We studied H. influenzae strains isolated in Spanish hospitals and, particularly, in one of the largest hospitals in the country. We found no increase in the incidence of H. influenzae type f in spite of there having been thorough vaccination against H. influenzae type b in Spain. Therefore, our data would not support the hypothesis of H. influenzae type f replacement after H. influenzae type b vaccination. Our results suggest that H. influenzae type f is an opportunistic pathogen. It causes infections in patients with serious underlying respiratory and liver pathologies or with impaired immunity. It is not a primary pathogen, as H. influenzae type b was in the pre-vaccination era.2 However, as in three of our clinical cases, occasional primary severe infections, such as meningitis or septicaemia, may appear in healthy children.

It has been suggested that invasive serotype a H. influenzae infections in children may be emerging as a consequence of molecular acquisition of a virulence genotype from H. influence type b.25 We found no such mechanism in our H. influenzae type f isolates since none—whether from invasive or non-invasive infections, isolated from children or adults—possessed the IS1016-bexA deletion.

Omikunle et al.35 studied 20 H. influenzae type f isolates by PFGE and found all but one to belong to a single clonotype. Our strains were also closely related since 91.6% of the strains had a genetic distance <=12%.

In summary, we studied the antibiotic susceptibility and clinical and epidemiological characteristics of 49 consecutive clinical isolates of H. influenzae type f. A high proportion of isolates were resistant to single and multiple antibiotics. Most strains were isolated from adults with severe underlying predisposing conditions, although cases of sepsis and meningitis may appear in children. The Spanish H. influenzae type f bacterial population is clonally related.


    Acknowledgements
 
This work was supported by a fellowship of the Instituto Carlos III (Reference 02/16), and the Autonomous Community of Madrid (Reference 08.2/0007/2001 1).

We are very grateful to Enrique Moguel for his technical assistance.

Members of the Spanish Study Group for H. influenzae type f: A. Carvajal (Hospital La Paz, Madrid), C. Miranda (Hospital Virgen de Las Nieves, Granada), J. L. Hernández (Hospital de Cruces, Barakaldo), A. Barrio (Hospital de Alcorcón, Alcorcón), R. Cantón and E. Loza (Hospital Ramón y Cajal, Madrid), D. Fontanals (Corporació Sanitaria Parc Taulí, Sabadell), J. L. Gómez-Garcés (Hospital de Móstoles, Móstoles), C. Rodriguez-Avial (Hospital Clínico San Carlos, Madrid), F. Brezmes and I. García (Hospital Virgen de La Concha, Zamora), C. Martí (Hospital General de Granollers, Granollers), F. Calbo (Hospital Infantil Carlos Haya, Málaga), M. Salvadó (Laboratori de Referència de Catalunya), J. M. Garcés (Hospital del Mar, Barcelona), R. Enriquez (Hospital Carlos III, Madrid), P. A. Carrero (Hospital General de Segovia, Segovia), C. García (Hospital Clínico Universitario "Lozano Blesa", Zaragoza)


    Footnotes
 
* Corresponding author. Tel: +34-91-822-36-50; Fax: +34-91-509-79-66; E-mail: jcampos{at}isciii.es Back

§ Members of the Spanish Study Group for H. influenzae type f are listed in the Acknowledgements. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1 . Campos, J. & Sáez-Nieto, J. A. (2001). Gram-negative infections: Haemophilus and other clinically relevant Gram negative coccobacilli. In Laboratory Diagnosis of Bacterial Infections (Cimolai, N., Ed.), pp. 557–80. Marcel Dekker, New York, NY, USA.

2 . Centers for Disease Control and Prevention. (2002). Progress toward elimination of Haemophilus influenzae type b invasive disease among infants and children—United States, 1998–2000. Morbidity and Mortality Weekly Report 51, 234–7.[Medline]

3 . Peltola, H. (2000). Worldwide Haemophilus influenzae type b diseases at the begininig of the 21st century: global analysis of the disease burden 25 years after use of the polysacharide vaccine and a decade after the advent of conjugates. Clinical Microbiology Reviews 13, 302–17.[Abstract/Free Full Text]

4 . Campos, J. (2001). Haemophilus influenzae: from the post-vaccination era to antibiotic resistance. Clinical Microbiology and Infection 7, 287–90.[CrossRef][ISI][Medline]

5 . Ward, J. I. (1996). Editorial response: Invasive infections due to Haemophilus influenzae serotype f (Hif)- Is Hif an emerging pathogen? Clinical Infectious Diseases 22, 1077–8.[ISI][Medline]

6 . Centers for Disease Control and Prevention. (2002). Serotyping discrepancies in Haemophilus influenzae type b disease—United States, 1998–1999. Morbidity and Mortality Weekly Report 51, 706–7.[Medline]

7 . LaClaire, L. L., Tondella, M. L. C., Beall, D. S. et al. (2003). Identification of Haemophilus influenzae serotypes by standard slide agglutination serotyping and PCR-based capsule typing. Journal of Clinical Microbiology 41, 393–6.[Abstract/Free Full Text]

8 . Urwin, G., Krohn, J. A., Deaver-Robinson, K. et al. (1996). Invasive disease due to Haemophilus influenzae serotype f: clinical and epidemiologic characteristics in the H. influenzae serotype b vaccine era. The Haemophilus influenzae Study Group. Clinical Infectious Diseases 22, 1069–76.[ISI][Medline]

9 . Waggoner-Fountain, L. A., Hendley, J. O., Cody, E. J. et al. (1995). The emergence of Haemophilus influenzae types e and f as significant pathogens. Clinical Infectious Diseases 21, 1322–4.[ISI][Medline]

10 . Chusid, M. J., Schneider, J. P., Thometz, J. G. et al. (1992). Osteomyelitis and septic arthritis caused by Haemophilus influenzae type f in a young girl. Diagnostic Microbiology and Infectious Diseases 15, 157–9.[CrossRef][ISI][Medline]

11 . Dworzack, D. L., Blessing, L. D., Hodges, G. R. et al. (1978). Haemophilus influenzae type F pnemonia in adults. American Journal of Medical Sciences 275, 87–91.[ISI]

12 . Glatman-Freedman, A. & Litman, N. (1996). Septic arthritis caused by an unusual type of Haemophilus influenzae. Journal of Infection 32, 143–5.[ISI][Medline]

13 . Gonzalez, M., del Pino, A., García, F. J. et al. (2000). Meningitis due to Haemophilus influenzae type f. Anales Españoles de Pediatría 53, 369–71.[Medline]

14 . Greene, G. R. (1978). Meningitis due to Haemophilus influenzae other than type b: case report and review. Pediatrics 62, 1021–5.[Abstract]

15 . Klein, B. L., Boxerbaum, B. & Aronoff, S. C. (1985). Haemophilus influenzae type f meningitis in an adolescent. Pediatric Emergency Care 1, 145–6.[Medline]

16 . Pincus, D. R. & Robson, J. M. (1998). Meningitis due to Haemophilus influenzae type f. Journal of Paediatric Child Health 34, 95–6.[CrossRef][ISI]

17 . Ratka, A. & Erramousque, J. (2001). Intramuscular ceftriaxone in the treatment of childhood meningitis due to Haemophilus influenzae type f. Annals of Pharmacotherapy 35, 36–40.[Abstract/Free Full Text]

18 . Slater, L. N., Guarnaccia, J., Makintubee, S. et al. (1990). Bacteremic disease due to Haemophilus influenzae capsular type f in adults: report of five cases and review. Reviews of Infectious Diseases 12, 628–35.[ISI][Medline]

19 . Wagener, W. C., Myerowitz, R. L. & Dulabon, G. M. (1981). Lethal meningoencephalitis and septicemia caused by Haemophilus influenzae type f in an adult with multiple myeloma. Journal of Clinical Microbiology 14, 695–6.[ISI][Medline]

20 . Yagupsky, P. & Rosenthal, D. (1998). Meningitis due to a strain of Haemophilus influenzae type f with intermediate susceptibility to ampicillin. Journal of Infection 16, 204–5.

21 . Zacharisen, M. C., Wartters, S. K. & Edwards, J. (2003). Rapidly fatal Haemophilus influenzae serotype f sepsis in a healthy child. Journal of Infection 46, 194–6.[CrossRef][ISI][Medline]

22 . Falla, T. J., Crook, D. W. M., Brophy, L. N. et al. (1994). PCR for capsular typing of Haemophilus influenzae. Journal of Clinical Microbiology 32, 2382–6.[Abstract]

23 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.

24 . Azemun, P., Stull, T., Roberts, M. et al. (1981). Rapid detection of chloramphenicol resistance in Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 20, 168–70.

25 . Adderson, E. E., Byington, C. L., Spencer, L. et al. (2001). Invasive serotype a Haemophilus influenzae infections with a virulence genotype resembling Haemophilus influenzae type b: emerging pathogen in the vaccine era? Pediatrics 108, E18.

26 . Leaves, N. I., Dimopoulou, I., Hayes, I. et al. (2000). Epidemiological studies of large plasmids in Haemophilus. Journal of Antimicrobial Chemotherapy 45, 599–604.[Abstract/Free Full Text]

27 . Tenover, F. C., Arbeit, R. D., Goering, R. V. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Free Full Text]

28 . De Andrade, A. L., Brandileone, M. C., Di Fabio, J. L. et al. (2001). Haemophilus influenzae resistance in Latin America: systematic review of surveillance data. Microbial Drug Resistance 7, 403–11.[CrossRef][ISI][Medline]

29 . Jones, M. E., Karlowsky, J. A., Blosser-Middleton, R. et al. (2002). Apparent plateau in ß-lactamase production among clinical isolates of Haemophilus influenzae and Moraxella catharralis in the United States: results of the LIBRA Surveillance initiative. International Journal of Antimicrobial Agents 19, 119–23.[CrossRef][ISI][Medline]

30 . Marco, F., García de Lomas, J., García-Rey, C. et al. (2001). Antimicrobial susceptibilities of 1,730 Haemophilus influenzae respiratory tract isolates in Spain in 1998–1999. Antimicrobial Agents and Chemotherapy 45, 3226–8.[Abstract/Free Full Text]

31 . Campos, J., García-Tornel, S. & Sanfeliu, I. (1984). Susceptibility studies of multiply resistant Haemophilus influenzae isolated from pediatric patients and contacts. Antimicrobial Agents and Chemotherapy 25, 706–9.

32 . Campos, J., Román, F., Pérez-Vázquez, M. et al. (2003) Infections due to Haemophilus influenzae type E: microbiological, clinical and epidemiological features. Clinical Infectious Diseases 37, 841–5.[CrossRef][ISI][Medline]

33 . Campos, J., Chanyangam, M., deGroot, R. et al. (1989). Genetic relatedness of antibiotic resistance determinants in multiply resistant Haemophilus influenzae. Journal of Infectious Diseases 60, 810–7.

34 . Levy, J., Verhaegen, G., De Mol, P. et al. (1993). Molecular characterization of resistance plasmids in epidemiologically unrelated strains of multiresistant Haemophilus influenzae. Journal of Infectious Diseases 168, 177–87.[ISI][Medline]

35 . Omikunle, A., Takahashi, S., Ogilvie, C. L. et al. (2002). Limited genetic diversity of recent invasive isolates of non-serotype b encapsulated Haemophilus influenzae. Journal of Clinical Microbiology 40, 1264–70.[Abstract/Free Full Text]