Haemophilus haemolyticus is a human commensal bacterium that colonizes the respiratory tract (
15).
H. haemolyticus is closely related to the human pathogen
Haemophilus influenzae but has rarely been reported to cause invasive disease.
H. haemolyticus and H. influenzae differ from other Haemophilus species because they require hemin (X factor) and NAD (V factor) for growth. H. haemolyticus can easily be distinguished from encapsulated H. influenzae because H. influenzae isolates produce one of the six structurally distinct capsules that can be easily determined by slide agglutination assay, whereas H. haemolyticus has never been shown to produce a capsule. However, due to the high similarity in morphology, biochemistry, and genetics between H. haemolyticus and nonencapsulated or nontypeable H. influenzae, distinguishing the two by standard microbiology methods has been challenging.
The presence of one or more of the following four features has been used for differentiating
H. haemolyticus from nontypeable
H. influenzae. (i)
H. haemolyticus induces clear β-hemolysis on horse blood agar. However, the hemolytic activity may be lost due to multiple subcultures or other unknown mechanisms, and nonhemolytic
H. haemolyticus strains are often misidentified as nontypeable
H. influenzae (
13,
15–
18). (ii) More than 90% of
H. haemolyticus strains produce hydrogen sulfide (H
2S); a small proportion of
H. influenzae biotype IV strains produce H
2S (
8,
10,
19). Therefore, production of H
2S cannot unambiguously distinguish
H. haemolyticus from
H. influenzae. (ii)
H. influenzae can be distinguished by PCR assays detecting the genes coding for protein D (
hpd) and the fuculose kinase gene (
fucK); a negative PCR result for both
hpd and
fucK genes is a strong indication of
H. haemolyticus (
17,
18,
23,
24). (iv) 16S rRNA gene sequences of
H. haemolyticus and
H. influenzae form different phylogenetic clusters. Recent studies have demonstrated that use of both phenotypic and genotypic assays improves identification of
H. influenzae and
H. haemolyticus isolated from asymptomatic carriers (
23).
To determine whether
H. haemolyticus invasive disease has been overlooked due to its high similarity to nontypeable
H. influenzae, we characterized isolates from cases reported to be caused by nontypeable
H. influenzae that were submitted to the CDC through routine surveillance in 2010 or through a population-based Active Bacterial Core surveillance (ABCs) program (
http://www.cdc.gov/abcs/index.htm) in 1999, 2000, 2009, and 2010 in the United States.
A total of 161 isolates from 2009 to 2010 and 213 isolates from 1999 to 2000 were characterized using phenotypic assays, including Kovac's oxidase test, API NH strips (bioMérieux; Durham, NC), hydrogen sulfide (H
2S) test strips (Sigma-Aldrich, Allentown, PA), and
Haemophilus ID Quad plates (Remel, Lenexa, KS). These isolates were further characterized using the
hpd and
fucK PCR assays that detect internal regions of the two genes (
9,
17,
18). All isolates showed typical
H. influenzae colony morphology, were positive for oxidase, required heme and NAD factors for growth, and produced hydrogen sulfide (H
2S). The isolates from cases 1 to 5 showed hemolytic activity on
Haemophilus ID Quad plates; other isolates (cases 6 and 7) were nonhemolytic. All seven isolates were negative for
hpd and
fucK by specific PCR assays.
16S rRNA gene sequencing was conducted on the seven isolates as described previously (
20). Phylogenetic and molecular evolutionary analyses of 16S rRNA gene sequences were conducted using MEGA version 4 using the neighbor-joining method (
22). Bootstrap numbers are from 1,000 replicates. 16S rRNA gene sequences of these isolates were compared with those of 10
H. haemolyticus isolates, 18
H. influenzae isolates, and 10 isolates representing
Haemophilus species other than
H. haemolyticus and
H. influenzae that were published in GenBank and the Ribosomal Database Project (
http://rdp.cme.msu.edu/) (
20). As shown in , isolates of the same species generally formed a group. The organisms from cases 1 to 7 were grouped with other known
H. haemolyticus but not
H. influenzae strains. Based on the profile of these phenotypic and genotypic laboratory tests, the seven isolates from cases 1 to 7 were identified as
H. haemolyticus. Genome sequences of isolates from case 6 (AFQR00000000) and 7 (AFQQ00000000) have recently been published (
9). The
fucK gene is missing, but the
hpd gene is present in these two isolates, suggesting that the PCR target region of the
hpd gene is species specific.
All seven patients infected with H. haemolyticus had different symptoms and presentations (). Two of the seven H. haemolyticus cases were from California and Oregon in 1999 and five cases were from Connecticut, Georgia, Minnesota, Illinois, and Texas in 2009 to 2010. All seven patients were hospitalized with no deaths. Isolates were recovered from 5 blood, 1 synovial fluid, and 1 pancreatic specimen (). Five of the seven isolates were submitted to the CDC through the ABCs program. The other two isolates from Illinois and Texas were collected from routine surveillance and submitted for confirmation of species identification.
| Table 1Summary of clinical and laboratory results for the seven casesb |
Haemophilus species other than
H. influenzae and
H. ducreyi are not common human pathogens (
1,
21). Only a few bacteremia cases caused by non-
H. influenzae Haemophilus species have been reported (
3,
6). Two endocarditis cases caused by
H. haemolyticus were reported in 1923 and in 1933 (
5,
14). Our clinical and laboratory findings indicate that
H. haemolyticus is associated with invasive clinical disease. Six patients had underlying medical conditions or recent surgical procedures that could have compromised their immune systems, potentially increasing their risk for invasive
H. haemolyticus infection.
H. haemolyticus may cause disease more as an opportunistic pathogen. Little is known about the mechanisms of
H. haemolyticus pathogenicity. Capsule is considered a crucial virulence factor for many bacterial pathogens, such as
H. influenzae (
11); however,
H. haemolyticus has not been reported to produce capsule.
H. influenzae outer membrane proteins and lipopolysaccharide of
H. influenzae play important roles in bacterial colonization and evasion of host defenses (
7). Because of the high frequency of horizontal gene transfer among and between
Haemophilus species, it is conceivable that the homologues of
H. influenzae virulent factors may be present in
H. haemolyticus and play a role in
H. haemolyticus pathogenicity. Future work is necessary to define the virulent determinants of
H. haemolyticus and the role of hemolysin as a virulence factor.
Our study also indicates that molecular tools are useful for discriminating
H. haemolyticus from nontypeable
H. influenzae when the phenotypic assays are unable to provide conclusive results (
23). Detection of the two
H. haemolyticus cases from 1999 suggests that disease caused by
H. haemolyticus has been historically overlooked;
H. haemolyticus cases have previously been misidentified as nontypeable
H. influenzae cases due to the lack of proper detection methods. In addition to the
hpd and
fucK genes, other biomarker genes have been proposed to differentiate
H. haemolyticus from
H. influenzae, such as the
H. influenzae adherence and penetration protein gene
hap, the [Cu, Zn]superoxide dismutase gene
sodC, the immunoglobulin A protease gene
iga, and the outer membrane protein P6 (
4,
13,
16–
18,
24). The
hpd and
iga genes are more reliable than the
fucK,
hap,
sodC, and protein P6 genes for distinguishing the two organisms (
2,
4,
12–
14,
23,
24). 16S rRNA gene sequencing is used for identification of bacterial species (
20), but the low bootstrap value () has suggested that single gene sequences may not be able to phylogenetically discriminate
H. haemolyticus and
H. influenzae. McCrea et al. have shown that concatenated sequences of multiple genes, including the 16S rRNA gene,
adk (adenylate kinase gene),
pgi (glucose-6-phosphate isomerase gene),
recA (recombination protein gene), and
infB (translation initiation factor 2 gene), separate the two species into distinct clusters (
13). As no single trait is able to unequivocally discriminate
H. haemolyticus and
H. influenzae, a testing scheme that includes reliable molecular techniques such as the
hpd- and
iga-based PCR assays can be used in combination with standard microbiological methods in order to improve the identification of
H. haemolyticus.
a Xin Wang,a Elizabeth C. Briere,a Lee S. Katz,b Amanda C. Cohn,a Thomas A. Clark,a Nancy E. Messonnier,a and Leonard W. Mayera
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