Insights into the population structure and pan-genome of Haemophilus influenzae.

The human-restricted bacterium Haemophilus influenzae is responsible for respiratory infections in both children and adults. While colonization begins in the upper airways, it can spread throughout the respiratory tract potentially leading to invasive infections. Although the spread of H. influenzae serotype b (Hib) has been prevented by vaccination, the emergence of infections by other serotypes as well as by non-typeable isolates (NTHi) have been observed, prompting the need for novel prevention strategies. Here, we aimed to study the population structure of H. influenzae and to get some insights into its pan-genome. We studied 305H. influenzae strains, enrolling 217 publicly available genomes, as well as 88 newly sequenced H. influenzae invasive strains isolated in Portugal, spanning a 24-year period. NTHi isolates presented a core-SNP-based genetic diversity about 10-fold higher than the one observed for Hib. The analysis of key factors involved in pathogenesis, such as lipooligosaccharides, hemagglutinating pili and High Molecular Weight-adhesins, suggests that NTHi shape its virulence repertoire, either by acquisition and loss of genes or by SNP-based diversification, likely towards host immune evasion and persistence. Discreet NTHi subpopulations structures are proposed based on core-genome supported with 17 candidate genetic markers identified in the accessory genome. Additionally, this study provides two bioinformatics tools for in silico rapid identification of H. influenzae serotypes and NTHi clades previously proposed, obviating laboratory-based demanding procedures. The present study constitutes an important genomic framework that could lay way for future studies on the genetic determinants underlying invasiveness and disease and population structure of H. influenzae.

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

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 ß-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 ß-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.

Antimicrobial resistance and epidemiological study of Haemophilus influenzae strains isolated in Portugal. The Multicentre Study Group.

In the course of a multicentric surveillance study, nine laboratories sent 375 isolates of Haemophilus influenzae to the Sector de Resistência aos Antibióticos (SRA) from the National Institute of Health in Lisbon, between 1 January and 31 December 1992. The majority of the H. influenzae isolates were from the respiratory tract (84.8%); only 5.1% were of invasive origin. Overall resistance for ampicillin was 11.7%, tetracycline 3.7%, and chloramphenicol 2.4%. All isolates tested were fully susceptible to cefotaxime, ceftriaxone, ciprofloxacin and rifampicin. Multiresistance was rare, occurring only in 2.4% of the isolates, although 50% of the ampicillin resistant strains had at least one additional resistance marker. Forty two isolates (11.2%) produced a TEM-1 type beta-lactamase, as shown by isoelectric focusing. beta-lactamase production was not detected in two of the ampicillin resistant strains. Fifteen of the 42 beta-lactamase producing strains (35.7%) contained detectable DNA plasmid: nine harboured large plasmids with an apparent molecular mass of 45 or 54 kb depending on their resistance phenotype and six harboured a small plasmid of 5 kb. In order to study transfer of resistance in both ampicillin and multiresistant strains conjugation experiments were performed for 14 isolates, seven of which harboured a large plasmid and seven had no detectable plasmid DNA. All 14 transferred their resistance phenotype but only a single large plasmid could be demonstrated in ten transconjugants. Restriction endonuclease analysis of plasmids from six representative transconjugants, isolated in different hospitals, revealed that there was no dissemination of a single R plasmid, which suggests an independent process of acquisition of resistance genes.

[Beta-lactamase-producing Haemophilus influenzae isolated in Portugal, 1989-1992]. / Haemophilus influenzae producteurs de bêtalactamase, isolés au Portugal de 1989-1992.

Within the framework of a national multicentric study between 1989 and 1992, 118 strains of betalactamase producing Haemophilus influenzae were isolated. Biotyping demonstrated the predominance of biotypes I, II and III, with 22, 36 and 24% of the strains, respectively. Encapsulated strains accounted for 13% of the total; all, but one, were serotype b. The antimicrobial susceptibility test (dilution method) of the 118 ampicillin--resistant strains showed: 33.9% resistance to tetracycline, 29.7% to chloramphenicol, 10.2% to erythromycin, 9.3% to trimethoprim, 0.8% to rifampicin, and 29.7% of multiresistance. All strains were susceptible to augmentin, cefotaxime, ceftriaxone and ceprofloxacin. Ninety strains were screened for resistant plasmids. A large plasmid (30-50 Mdal) was isolated in 38.9% of the strains and a small plasmid (3-4.4 Mdal) in 10%. No plasmid was found in 51% of the strains. Isoelectric focusing of 54 beta-lactamases showed that all were type TEM-1 (pI = 5.4), with the exception of one, which was type TEM-2 (pI = 5.6).