haemophilus haemolyticus causing clinical disease raydel anderson*1, xin wang1, elizabeth c
TRANSCRIPT
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Haemophilus haemolyticus causing clinical disease 1
Raydel Anderson*1, Xin Wang1, Elizabeth C. Briere1, Lee S. Katz2, Amanda C. Cohn1, 2
Thomas A. Clark1, Nancy E. Messonnier1, Leonard W. Mayer1 3
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Running title: Haemophilus haemolyticus cases in the United States 5
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1Meningitis and Vaccine Preventable Disease Branch, Division of Bacterial Diseases, National 7
Center for Infectious and Respiratory Diseases, Centers for Disease Control and Prevention, 8
Atlanta, Georgia 30333 9
2 Enteric Diseases Laboratory Branch, Division of Foodborne, Waterborne, and Environmental 10
Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease 11
Control and Prevention, Atlanta, Georgia 30333 12
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*Corresponding author. Mailing address: Centers for Disease Control and Prevention, 14
NCIRD/DBD/MVPDB, 1600 Clifton Rd NE, MS D-11, Atlanta, Georgia 30033, USA. Phone: 15
404-639-5490. Fax: 404-639-4421. Email: [email protected] 16
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Note: The findings and conclusions in this report are those of the authors and do not 20
necessarily represent the official position of the CDC. 21
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.06575-11 JCM Accepts, published online ahead of print on 9 May 2012
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ABSTRACT 24
We report seven cases of Haemophilus haemolyticus invasive disease detected in the 25
United States, which were previously misidentified as non-typeable Haemophilus influenzae 26
(Hi). All cases had different symptoms and presentations. Our study suggests that a testing 27
scheme that includes reliable PCR assays and standard microbiological methods should be used 28
in order to improve H. haemolyticus identification. 29
TEXT 30
Haemophilus haemolyticus is a human commensal bacterium that colonizes the 31
respiratory tract (15). H. haemolyticus is closely related to the human pathogen Haemophilus 32
influenzae but has rarely been reported to cause invasive disease. 33
H. haemolyticus and H. influenzae differ from other Haemophilus species because they 34
require hemin (X factor) and nicotinamide adenine dinucleotide (NAD, V factor) for growth. 35
H. haemolyticus can be easily distinguished from encapsulated H. influenzae because H. 36
influenzae isolates produce one of the six structurally distinct capsules that can be easily 37
determined by slide agglutination assay, whereas, H. haemolyticus has never been shown to 38
produce a capsule. However, due to the high similarity in morphology, biochemistry and 39
genetics between H. haemolyticus and non-encapsulated or non-typeable H. influenzae, 40
distinguishing the two by standard microbiology methods has been challenging. 41
The presence of one or more of the following four features has been used for 42
differentiating H. haemolyticus from non-typeable H. influenzae: 1) H. haemolyticus induces 43
clear (beta)-hemolysis on horse blood agar. However, the hemolytic activity may be lost due to 44
multiple subcultures or other unknown mechanisms, and non-hemolytic H. haemolyticus are 45
often misidentified as non-typeable H. influenzae (13, 15-18). 2) More than 90% of H. 46
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haemolyticus produce hydrogen sulfide (H2S); a small proportion of H. influenzae biotype IV 47
produces H2S (8, 10, 19). Therefore, production of H2S cannot unambiguously distinguish H. 48
haemolyticus from Hi. 3) H. influenzae can be distinguished by PCR assays detecting the 49
genes coding for protein D (hpd) and the fuculose kinase (fucK); A negative PCR result for 50
both hpd and fucK genes is a strong indication of H. haemolyticus (17, 18, 23, 24). 4) 16S 51
rRNA gene sequences of H. haemolyticus and H. influenzae form different phylogenetic 52
clusters. Recent studies have demonstrated that use of both phenotypic and genotypic assays 53
improves identification of H. influenzae and H. haemolyticus isolated from asymptomatic 54
carriers (23). 55
To determine whether H. haemolyticus invasive disease has been overlooked due to its 56
high similarity to non-typeable H. influenzae, we characterized isolates from cases reported to 57
be caused by non-typeable H. influenzae that were submitted to CDC through routine 58
surveillance in 2010 or through a population-based Active Bacterial Core surveillance (ABCs) 59
program (www.cdc.gov/ncidod/DBMD/abcs/) in 1999, 2000, 2009, and 2010 in the United 60
States. 61
A total of 161 isolates from 2009-2010 and 213 isolates from 1999-2000 were 62
characterized using phenotypic assays including Kovac’s oxidase test, API® NH strips 63
(bioMerieux, Durham, North Carolina), hydrogen sulfide (H2S) test strips (Sigma-Aldrich; 64
Allentown, PA), and Haemophilus ID Quad plates (Remel, Lenexa, Kansas). These isolates 65
were further characterized using the hpd and fucK PCR assays that detect internal regions of 66
the two genes (9, 17, 18). All isolates showed typical H. influenzae colony morphology, were 67
positive for oxidase, required heme and NAD factors for growth, and produced hydrogen 68
sulfide (H2S). The isolates from cases 1-5 showed hemolytic activity on Haemophilus ID Quad 69
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plates, other isolates (cases 6 and 7) were non-hemolytic. All seven isolates were negative for 70
hpd and fucK by specific PCR assays. 71
16S rRNA gene sequencing was conducted on the seven isolates as described 72
previously (20). Phylogenetic and molecular evolutionary analyses of 16S rRNA gene 73
sequences were conducted using MEGA version 4 using the neighbor-joining method (22). 74
Bootstrap numbers are from 1000 replicates. 16S rRNA gene sequences of these isolates were 75
compared with that of 10 H. haemolyticus isolates, 18 H. influenzae isolates, and 10 isolates 76
representing Haemophilus species other than H. haemolyticus and H. influenzae that were 77
published in GenBank and Ribosomal Database Project (http://rdp.cme.msu.edu/) (20). As 78
shown in Fig. 1, isolates of the same species generally formed a group. Cases 1-7 were 79
grouped with other known H. haemolyticus, but not H. influenzae. Based on the profile of 80
these phenotypic and genotypic laboratory tests, the seven isolates from case 1-7 were 81
identified as H. haemolyticus. Genome sequences of isolates from case 6 (AFQR00000000) 82
and 7 (AFQQ00000000) have recently been published (9). The fucK gene is missing, but the 83
hpd gene is present in these two isolates, suggesting the PCR target region of the hpd gene is 84
species-specific. 85
All seven patients infected with H. haemolyticus had different symptoms and 86
presentations (Table 1). Two of the seven H. haemolyticus cases were from California and 87
Oregon in 1999 and five cases were from Connecticut, Georgia, Minnesota, Illinois and Texas 88
in 2009-2010. All seven patients were hospitalized with no deaths. Isolates were recovered 89
from 5 blood, 1 synovial fluid, and 1 pancreatic specimen (Table 1). Five of the seven isolates 90
were submitted to CDC through ABCs. The other two isolates from IL and TX were collected 91
from routine surveillance and submitted for confirmation of species identification. 92
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Haemophilus species other than H. influenzae and H. ducreyi are not common human 93
pathogens (1, 21). Only a few bacteremia cases caused by non- H. influenzae, Haemophilus 94
species have been reported (3, 6). Two endocarditis cases caused by H. haemolyticus were 95
reported in 1923 and in 1933 (5, 14). Our clinical and laboratory findings indicate that H. 96
haemolyticus is associated with invasive clinical disease. Six patients had underlying medical 97
conditions or recent surgical procedures that could have compromised their immune systems, 98
potentially increasing their risk for invasive H. haemolyticus infection. H. haemolyticus may 99
cause disease more as an opportunistic pathogen. Little is known about the mechanisms of H. 100
haemolyticus pathogenicity. Capsule is considered a crucial virulence factor for many bacterial 101
pathogens such as H. influenzae (11); however H. haemolyticus has not been reported to 102
produce capsule. H. influenzae outer membrane proteins and lipopolysaccharide of H. 103
influenzae play important roles in bacterial colonization and evasion of host defenses (7). 104
Because of the high frequency of horizontal gene transfer among and between Haemophilus 105
species, it is conceivable that the homologues of H. influenzae virulent factors may be present 106
in H. haemolyticus and play a role in H. haemolyticus pathogenicity. Future work is necessary 107
to define the virulent determinants of H. haemolyticus, and the role of hemolysin as a virulence 108
factor. 109
Our study also indicates that molecular tools are useful for discriminating H. 110
haemolyticus from non-typeable H. influenzae when the phenotypic assays are unable to 111
provide conclusive results (23). Detection of the two H. haemolyticus cases from 1999 112
suggests that disease caused by H. haemolyticus has been historically overlooked; H. 113
haemolyticus cases have previously been misidentified as non-typeable H. influenzae cases due 114
to the lack of proper detection methods. In addition to the hpd and fucK genes, other biomarker 115
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genes have been proposed to differentiate H. haemolyticus from H. influenzae such as the H. 116
influenzae adherence and penetration protein gene hap, the [Cu, Zn]-superoxide dismutase 117
gene sodC, and the immunoglobulin A protease gene iga, and the outer membrane protein P6 118
(4, 13, 16-18, 24). The hpd and iga genes are more reliable than the fucK, hap, sodC and 119
protein P6 genes for distinguishing the two organisms (2, 4, 12-14, 23, 24). 16S rRNA gene 120
sequencing is used for identification of bacterial species (20), but the low bootstrap value 121
(Figure 1) has suggested that single gene sequences may not be able to phylogenetically 122
discriminate H. haemolyticus and H. influenzae. McCrea et al. has shown concatenated 123
sequences of multiple genes including16S rRNA gene, adk (adenylate kinase gene), pgi 124
(glucose-6-phosphate isomerase gene), recA (recombination protein gene) and infB (translation 125
initiation factor 2 gene) separate the two species into distinct clusters (13). As no single trait is 126
able to unequivocally discriminate H. haemolyticus and H. influenzae, a testing scheme that 127
includes reliable molecular techniques such as the hpd- and iga- based PCR assays can be used 128
in combination with standard microbiological methods in order to improve the identification of 129
H. haemolyticus. 130
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ACKNOWLEDGEMENT 132
We thank Active Bacterial Core surveillance team, Roman Golash from Illinois Department of 133
Health, and the Texas Department of State Health Services for providing case information and 134
isolates. We thank the CDC Special Bacteriology Reference Laboratory for assistance with 135
16S rRNA gene sequencing and Mary McCauley at CDC for her critical review. 136
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Table 1. Summary of clinical and laboratory results for the seven cases 217
Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Year of Isolation 1999 1999 2009 2009 2010 2010 2010 State CA OR CT MN GA IL TX Submission type ABCs ABCs ABCs ABCs ABCs Routine Routine Specimen type Blood Blood Blood Pancreas Blood Blood Synovial
fluid Sex Male Female female female female male male Age 50 65 47 63 6 19 60 Underlying condition(s) Metastatic
lung cancer, alcoholism
COPD, alcoholism, atherosclerotic cardiovascular disease, chronic heart failure
smoker, intravenous drug user, cirrhosis, alcoholism, Hepatitis C positive
diabetes mellitus, obesity
none gunshot wound
knee surgery
Syndrome bacteremia without focus
bacteremia pneumonia
bacteremia without focus
peritonitis syndrome
bacteremia without focus
bacteremia septic arthritis
Hospitalization length (days) 5 5 43 20 unknown 10+ unknown
NAD requirement + + + + + + + Heme requirement + + + + + + + Hemolysis + + + + + - -
H2S production + + + + + + +
hpd PCR negative negative negative negative negative negative1 negative1
fucK PCR negative negative negative negative negative negative2 negative2 Identification by 16S rRNA gene sequence
H. haemo- lyticus
H. haemo- lyticus
H. haemo- lyticus
H. haemo- lyticus
H. haemo- lyticus
H. haemo- lyticus
H. haemo- lyticus
1 The hpd gene is present in the isolates from case 6 and 7. 2 The fucK gene is absent in the isolates from case 6 and 7.
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Fig.1. Phylogenetic tree based on 16S rRNA gene sequences of different Haemophilus species 218
strains. The tree was generated using neighbor-joining in MEGA 4, with 1000 replicates and 219
shows the genetic relatedness of the isolates from the five cases to different Haemophilus 220
species. Hi and Hhae stand for H. influenzae and H. haemolyticus, respectively. The name of 221
each 16S rRNA gene sequence includes bacterial species, accession number and for H. 222
influenzae, the strain type number (Hi_AY613586_T9) for published sequences as well as a 223
case number (case1-7) for sequences in this study. All H. influenzae strains were isolated from 224
blood with the exception of AY613446, which was from CSF. The H. ducreyi strain was 225
isolated from a chancroid ulcer. The H. parainfluenzae strains AY365452 and AY365450 were 226
isolated from blood, M75081 and AY362908 are ATCC strains, and EU083530 an unpublished 227
source. The H. haemolyticus EU185350, EU185348, and EU185340 were invasive strains 228
from neonatal urogenital tracts. EU185345, EU185346, EU185339, EU185349, and 229
EU185354 were isolated from sputum or nasopharyngeal swabs from COPD patients and 230
M75045 is the type strain from NCTC. The H. paraphrohaemolyticus is the type strain from 231
NCTC and the H. parasius was from an unpublished source. 232
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