Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA

Abstract

Previous genetic analysis of Haemophilus influenzae strains isolated from genital and neonatal infections identified a group of biotype IV that constitutes a cryptic genospecies only distantly related to H. influenzae and H. haemolyticus. Small-subunit rRNA genes of two representative strains of this genital Haemophilus genospecies (strains 16N and 2406) were sequenced. The analysis indicated that these strains form a monophyletic unit with H. haemolyticus and H. influenzae biogroups Influenzae and Aegyptius and are more closely related to H. haemolyticus than to H. influenzae biogroups Influenzae and Aegyptius. 16S rRNA gene sequences were used to formulate primers for PCR-based identification of cryptic genital Haemophilus organisms. A 242-bp fragment was amplified from strains belonging to the genital Haemophilus genospecies but not from strains of 12 other Haemophilus species, including strains of H. influenzae biotype IV sensu stricto.

Comparison of the first whole genome sequence of ‘Haemophilus quentini’ with two new strains of ‘Haemophilus quentini’ and other species ofHaemophilus

“…Previous phylogenetic analysis of the sequenced 16S rRNA gene suggested that H. quentini was more closely related to H. haemolyticus than H. influenzae Quentin et al (1996). However, this was not fully determined beyond the initial 16S rRNA comparison.…”

“…Initially DNA-DNA hybridisation and restriction fragment length polymorphism identified that at least a subset of H. influenzae biotype IV may actually be distinct, but still related, albeit distantly, from both H. influenzae and Haemophilus haemolyticus Quentin et al (1993).Subsequent phylogenetic analysis of a partial sequence of the 16S rRNA gene of two strains of H. influenzae biotype IV found that a proportion of H. influenzae biotype IV were actually more closely related to H. haemolyticus than H. influenzae Quentin et al (1996) and unofficially renamed 'Haemophilus quentini', described as a "cryptic genospecies" since it cannot be distinguished phenotypically from H. influenzae Quentin et al (1996). Since this discovery, it has been identified as the cause of urinary tract infections in men Glover et al (2011) and neonatal bacteraemia through 16S rRNA sequencing Giufre et al (2015); Hubbard et al (2016); Mak et al (2005).…”

Reduced Levofloxacin Susceptibility and Tetracycline Resistance in a Clinical Isolate of Haemophilus quentini Identified by 16S rRNA Sequencing

“…Haemophilus strains isolated from neonatal and genital tract infections (4). Members of this species comprise part of the bacterial population identified as H. influenzae biotype IV in biochemical tests and were previously referred to as "a cryptic genospecies of Haemophilus" (2,11,12). They form a homogenous group with unique multilocus enzyme electrophoresis patterns, outer membrane protein profiles, and fimbrial protein gene sequences (2,9).…”

“…They form a homogenous group with unique multilocus enzyme electrophoresis patterns, outer membrane protein profiles, and fimbrial protein gene sequences (2,9). In 16S rRNA sequence analysis, they form a monophyletic unit with a closer relation to Haemophilus haemolyticus than to H. influenzae (12).…”

Letter to the Editor

“…Eight biotypes exist for each species, designated biotype I through biotype VIII (3-6). Despite having limited discriminatory power, when used in conjunction with other methods including newer genetic tools, biotyping may be helpful in the clinical laboratory to identify Haemophilus aegyptius (biotype III), which causes bacterial conjunctivitis 7, and Haemophilus quentini (biotype IV), which causes urogenital and neonatal infections (8).…”