High levels of multiple antibiotic resistance among 938 Haemophilus influenzae type b meningitis isolates from Cuba (1990–2002)

Isis Tamargo1, Kiomy Fuentes1, Alina Llop1, Jesús Oteo2 and José Campos2,*

1 Laboratorio Nacional de Referencia de Haemophilus, Instituto de Medicina Tropical ‘Pedro Kouri’, La Habana, Cuba; 2 Centro Nacional de Microbiología, Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Carretera Pozuelo a Majadahonda, 28220. Majadahonda, Madrid, Spain

Received 31 October 2002; returned 2 April 2003; revised 3 April 2003; accepted 29 June 2003


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Objectives: A national surveillance study to determine antimicrobial susceptibility in Haemophilus influenzae type b isolated from cerebrospinal fluid was carried out in Cuba from 1990 to 2002.

Methods: Susceptibility to ampicillin, co-amoxiclav, cefotaxime, ceftriaxone, co-trimoxazole, tetracycline, chloramphenicol and rifampicin was tested by the microdilution method according to the NCCLS guidelines.

Results: The 34 participating laboratories recovered 938 consecutive, non-identical isolates. All the isolates were retrieved from children aged <5 years. The mean number of isolates collected by year in the pre-vaccination era (1990–1998) was 93; after vaccination, 57 isolates were reported in 1999, 31 in 2000, four in 2001 and five in 2002. Resistance to ampicillin, co-trimoxazole, tetracycline and chloramphenicol was 46.3% (all ß-lactamase-positive), 51.3%, 33.2% and 44.0%, respectively. Ampicillin-resistant ß-lactamase-negative strains were not detected. All strains were susceptible to co-amoxiclav, cefotaxime, ceftriaxone and rifampicin. Ampicillin resistance was strongly associated with resistance to tetracycline, co-trimoxazole and chloramphenicol (P < 0.001). Multidrug resistance was present in 43.8% of isolates. The most prevalent phenotype was resistance to ampicillin/chloramphenicol/tetracycline/co-trimoxazole, which was detected in 29.2% of strains overall. An increase in the prevalence of resistance to these antibiotics was observed from 1990 to 2000 in the range 40.7%–54.8% for ampicillin, 40.1%–51.6% for chloramphenicol, 45.4%–58.1% for co-trimoxazole and 23%–45.2% for tetracycline.

Conclusions: In Cuba, the widespread vaccination against Haemophilus influenzae type b prevented a large number of meningitis cases in children caused by strains resistant to multiple antibiotics.

Keywords: Haemophilus influenzae type b, antimicrobial resistance, meningitis, conjugate vaccines


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Haemophilus influenzae type b (Hib) has been recognized as an important cause of pneumonia and meningitis. Before conjugate vaccines were available, it was one of the most prevalent bacterial pathogens to cause meningitis in children <5 years. With the widespread use of effective conjugate vaccines, the incidence of serotype b invasive infections has decreased drastically, although Hib remains a major public health problem, being responsible annually for more than 3 million cases of invasive disease worldwide.1

Since the 1970s, the prevalence of ampicillin resistance as a result of ß-lactamase production in H. influenzae has increased worldwide, reaching 20%–30% according to two studies performed both in Hib and non-capsulate strains.2,3 Resistance to other antimicrobial agents such as chloramphenicol, tetracycline and co-trimoxazole has also been reported. The great prevalence of multiresistance has been described in Hib isolates from Spain4 and Belgium.5

In this multicentre surveillance study, we describe the susceptibility of 938 strains of Hib from cerebrospinal fluid (CSF) isolated during 1990–2002 in 34 hospitals in Cuba.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Consecutive non-identical isolates of Hib obtained from CSF, collected during January 1990–October 2002 from 34 microbiology laboratories in Cuba, were included in this study. Strains were sent to a central laboratory (Laboratorio Nacional de Referencia de Haemophilus, Instituto de Medicina Tropical ‘Pedro Kouri’, La Habana, Cuba), where standard laboratory methods confirmed the identification. In addition, a slide agglutination test using type b-specific antiserum (Difco Laboratories, Detroit, MI, USA) was performed. ß-Lactamase production was determined by the chromogenic cephalosporin test with nitrocephin as substrate.

NCCLS guidelines6 were followed for susceptibility testing, which was conducted using a microdilution method in Haemophilus Test Medium Broth. The antibiotics tested were ampicillin (Sigma Chemical Co., St Louis, MO, USA), co-amoxiclav (Gautier-Bagó, La Habana, Cuba), cefotaxime (Sigma), ceftriaxone (Sigma), co-trimoxazole (Empresa Farmacéutica, La Habana, Cuba), tetracycline (Sigma), chloramphenicol (Merck, Barcelona, Spain) and rifampicin (Sigma). Final concentrations were ampicillin 0.06–256 mg/L, co-amoxiclav 0.25/0.12–8/4 mg/L (amoxicillin/clavulanic acid), cefotaxime 0.06–4 mg/L, ceftriaxone 0.06–4 mg/L, co-trimoxazole 0.25/4.75–8/152 mg/L, tetracycline 0.25–64 mg/L, chloramphenicol 0.25–64 mg/L and rifampicin 0.25–8 mg/L.

Following NCCLS recommendations,6 strains of H. influenzae ATCC 49766 and ATCC 49247 were used for quality control

Differences between ß-lactamase-negative and ß-lactamase-positive strains with respect to resistance to non-ß-lactam antibiotics, and differences in antimicrobial resistance by year, were assessed by the {chi}2 test. Association was determined by calculation of odds ratios (ORs) with 95% confidence intervals (CIs). The null hypothesis was rejected for values of P < 0.05. Statistical analyses were carried out using EPI-Info 2000 version 1.1.1.


    Results and discussion
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
The 34 participating laboratories collected 938 clinical, non-identical isolates of Hib obtained from meningitis patients during 1990–2002. All cases occurred in children <5 years.

MICs for the two control strains were always within recommended limits.6

Vaccine administration began in Cuba in 1999 in children born in that year. Before that date, the mean annual number of strains from CSF received in the Cuban Haemophilus Reference Laboratory during 1990–1998 was 93. After vaccination, there were 57 isolates reported in 1999, 31 in 2000, four in 2001 and five in 2002. A previous study reported that the incidence of Hib meningitis in children <5 years in Cuba, after conjugate vaccine administration, decreased from 17.3 cases per 100 000 inhabitants in 1998 to 7.6 cases per 100 000 inhabitants in 1999 (56.1%).7

The annual rate of evolution of resistance to antimicrobial agents in Hib isolates from CSF in Cuba is shown in Figure 1. The last 2 years are not included because of the small number of isolates. An increase in the prevalence of resistance to these antibiotics was observed from 1990 to 2000: from 40.7% to 54.8% for ampicillin; 40.1%–51.6% for chloramphenicol; 45.4%–58.1% for co-trimoxazole; 23%–45.2% for tetracycline. Only in the case of tetracycline resistance was the difference statistically significant (P=0.011; OR: 0.36; 95% CI: 0.15–0.87).



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Figure 1. Annual rate of evolution of resistance (%) to antimicrobial agents in H. influenzae type b isolates from CSF in Cuba from 1990 to 2000 (2001 and 2002 are not included because of the very low number of isolates).

 
Nearly half the isolates (434 of 938) were ß-lactamase producers (46.3%;). ß-Lactamase-negative ampicillin-resistant strains were not detected.

The prevalence of susceptibility to the antimicrobial agents tested, the range of MICs and MICs for 50% and 90% of strains (MIC50 and MIC90)—overall and according to ß-lactamase production—are shown in Table 1 A total of 434 (46.3%), 481 (51.3%), 413 (44%) and 311 (33.2%) strains were not susceptible to ampicillin, co-trimoxazole, chloramphenicol or tetracycline, respectively. Ampicillin resistance was strongly associated with resistance to tetracycline (P < 0.001; OR: 41.9; 95% CI: 25.6–69.3), to co-trimoxazole (P < 0.001; OR: 202.3; 95% CI: 109.6–379.1) and to chloramphenicol (P < 0.001; OR: 260.15; 95% CI: 142–482.4).


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Table 1. Susceptibility of H. influenzae type b from CSF to antimicrobial agents according to ß-lactamase production.
 
Multidrug resistance (defined as being non-susceptible to three or more classes of antibiotics) was present in 411 (43.8%) isolates. The pattern most frequently identified was resistance to ampicillin/chloramphenicol/tetracycline/co-trimoxazole (274 isolates, 29.2%), followed by resistance to ampicillin/chloramphenicol/co-trimoxazole (106 isolates, 11.3%). All multidrug-resistant isolates were non-susceptible to chloramphenicol, and 387 (94.2% of multiresistant strains) were non-susceptible to ampicillin and chloramphenicol. All ampicillin-resistant strains had at least one additional resistance marker. Of the four 2001 isolates, three were resistant to ampicillin/chloramphenicol/tetracycline/co-trimoxazole; and four out of five 2002 isolates presented resistance to ampicillin/chloramphenicol/tetracycline/co-trimoxazole.

No strains were resistant to co-amoxiclav, cefotaxime, ceftriaxone or rifampicin.

The production of ß-lactamase, as detected in this study, was one of the greatest reported anywhere in the world.2,3,8 Of 2100 invasive H. influenzae isolates from 15 Latin American countries during the same period, 450 (21.4%) were ß-lactamase producers.2 The production of this enzyme in clinical isolates in the USA has remained essentially unchanged at ~30% in recent years.3 Although there were important geographical variations, the overall prevalence of ß-lactamase production detected in a collaborative European study (The Alexander Project) was <12% in 1997–1998.8 By contrast, ß-lactamase production was 50% in Spanish invasive isolates of Hib.4

In a study carried out in La Habana city from 1992 to 1993, 22 of 55 (40%) Hib strains isolated from invasive and non-invasive infections produced ß-lactamase.9

In Cuba, the high levels of multidrug resistance were all from strains from CSF and from children aged <5 years. In contrast, the majority of published results include data of isolates of different types and from different sources.2,3

Antimicrobial resistance and cross-resistance is generally more frequent in Hib strains isolated from blood and CSF than from other sources.2,4 In a study carried out in Spain, 20 of 44 (45.4%) invasive Hib isolates were multiresistant, compared with 19 of 181 (10.5%) non-invasive isolates.4 In a multinational study in Latin America, ampicillin resistance was more prevalent in invasive than in non-invasive isolates, 21.9% versus 17.2%, respectively.2 However, in the same study co-trimoxazole resistance was more common in non-invasive than in invasive strains (41.9% versus 26.9%, respectively).8

Carriage of high-molecular-weight conjugative plasmids in Hib constitutes the genetic bases of resistance to ampicillin, chloramphenicol and tetracycline,5,10 which may explain the strong association between resistances to these antibiotics. However, in strains resistant to co-trimoxazole, the trimethoprim resistance determinant is chromosomal and not linked to the other antibiotic resistance determinants.10 It would appear that plasmid and chromosomal resistance have evolved independently.

The widespread use of effective vaccines against Hib generates a novel epidemiological situation. As we have shown in this study, vaccination campaigns against Hib have contributed to the drastic reduction in the number of Hib meningitis cases in children, as well as to the strong decrease in the number of multiple resistant Hib strains causing invasive diseases.

The excess of antimicrobial consumption in Cuba and in other parts of the world, which is probably more frequent in children than in adults, could explain the high rates of resistance in Hib and other pathogens. In addition, the presence of multidrug resistance may generate co-selection from treatment with only one of these antibiotics. Continued monitoring of susceptibility trends of H. influenzae, including typeable and non-typeable strains, will be required in order to decide on appropriate chemotherapy in the post-Hib vaccination era.


    Acknowledgements
 
This work was supported by Instituto de Medicina Tropical ‘Pedro Kouri’ and by the Instituto de Salud Carlos III, Ministry of Health, Spain (Reference MPY1145/02).


    Footnotes
 
* Corresponding author. Tel: +34-91-509-79-01; Fax: +34-91-509-79-66; E-mail: jcampos{at}isciii.es Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
1 . Peltola, H. (2000). Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. Clinical Microbiology Reviews 13, 302–17.[Abstract/Free Full Text]

2 . 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]

3 . 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 catarrhalis in the United States: results from the LIBRA Surveillance initiative. International Journal of Antimicrobial Agents 19, 119–23.[CrossRef][ISI][Medline]

4 . 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.[ISI][Medline]

5 . 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 Bacteriology 138, 584–97.

6 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

7 . Dickinson, F. O., Pérez, A. E., Galindo, M. A. et al. (2001). Impact of vaccination against Haemophilus influenzae type b in Cuba. Revista Panamericana de Salud Pública 10, 169–73.[Medline]

8 . Schito, G. C., Debbia, E. A. & Marchese, A. J. (2000). The evolving threat of antibiotic resistance in Europe: new data from the Alexander Project. Journal of Antimicrobial Chemotherapy 46, 3–9.[Abstract/Free Full Text]

9 . Llanes, R., Azahares, L. E., Perez, M. F. et al. (1996). Antimicrobial resistance in Haemophilus influenzae in the city of La Havana, Cuba. Revista Argentina de Microbiología 28, 17–21.[Medline]

10 . Campos, J., Chanyangam, M., deGroot, R. et al. (1989). Genetic relatedness of antibiotic resistance determinants in multiply resistant Haemophilus influenzae. Journal of Infectious Diseases 160, 810–7.[ISI][Medline]