1

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

4

Running title: Haemophilus haemolyticus cases in the United States 5

6

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

13

*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: rdo7@cdc.gov 16

17

18

19

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

22

23

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

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

2

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

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

3

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

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

4

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

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

5

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

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

6

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

131

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

137

REFERENCES 138

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

7

1. Albritton, W. L. 1989. Biology of Haemophilus ducreyi. Microbiological reviews 139

53:377-389. 140

2. Binks, M. J., B. Temple, L. A. Kirkham, S. P. Wiertsema, E. M. Dunne, P. C. 141

Richmond, R. L. Marsh, A. J. Leach, and H. C. Smith-Vaughan. 2012. Molecular 142

Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR 143

Screening Assays. PloS one 7:e34083. 144

3. Cardines, R., M. Giufre, M. L. Ciofi degli Atti, M. Accogli, P. Mastrantonio, and 145

M. Cerquetti. 2009. Haemophilus parainfluenzae meningitis in an adult associated 146

with acute otitis media. The new microbiologica 32:213-215. 147

4. Chang, A., D. G. Adlowitz, E. Yellamatty, and M. Pichichero. 2010. Haemophilus 148

influenzae outer membrane protein P6 molecular characterization may not differentiate 149

all strains of H. Influenzae from H. haemolyticus. Journal of clinical microbiology 150

48:3756-3757. 151

5. De Santo, D. A., and M. White. 1933. Hemophilus Hemolyticus Endocarditis. The 152

American journal of pathology 9:381-392 381. 153

6. Douglas, G. W., L. L. Buck, and C. Rosen. 1979. Liver abscess caused by 154

Haemophilus paraphrohaemolyticus. Journal of clinical microbiology 9:299-300. 155

7. Foxwell, A. R., J. M. Kyd, and A. W. Cripps. 1998. Nontypeable Haemophilus 156

influenzae: pathogenesis and prevention. Microbiol Mol Biol Rev 62:294-308. 157

8. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams. 1994. 158

Bergey’s Manual of Determinative Bacteriology. Chapter 5: Facultatively anaerobic 159

gram negative rods. 9th edition. 160

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

8

9. Jordan, I. K., A. B. Conley, I. V. Antonov, R. A. Arthur, E. D. Cook, G. P. Cooper, 161

B. L. Jones, K. M. Knipe, K. J. Lee, X. Liu, G. J. Mitchell, P. R. Pande, R. A. Petit, 162

S. Qin, V. N. Rajan, S. Sarda, A. Sebastian, S. Tang, R. Thapliyal, N. J. Varghese, 163

T. Ye, L. S. Katz, X. Wang, L. Rowe, M. Frace, and L. W. Mayer. 2011. Genome 164

sequences for five strains of the emerging pathogen Haemophilus haemolyticus. Journal 165

of bacteriology 193:5879-5880. 166

10. Kilian, M. 1976. A taxonomic study of the genus Haemophilus, with the proposal of a 167

new species. J Gen Microbiol 93:9-62. 168

11. Kroll, J. S. 1992. The genetics of encapsulation in Haemophilus influenzae. J Infect Dis 169

165 Suppl 1:S93-96. 170

12. McCrea, K. W., M. L. Wang, J. Xie, S. A. Sandstedt, G. S. Davis, J. H. Lee, C. F. 171

Marrs, and J. R. Gilsdorf. 2010. Prevalence of the sodC gene in nontypeable 172

Haemophilus influenzae and Haemophilus haemolyticus by microarray-based 173

hybridization. Journal of clinical microbiology 48:714-719. 174

13. McCrea, K. W., J. Xie, N. LaCross, M. Patel, D. Mukundan, T. F. Murphy, C. F. 175

Marrs, and J. R. Gilsdorf. 2008. Relationships of nontypeable Haemophilus 176

influenzae strains to hemolytic and nonhemolytic Haemophilus haemolyticus strains. 177

Journal of clinical microbiology 46:406-416. 178

14. Miller, C. P., and A. Branch. 1923. Subacute bacterial endocarditis due to a hemolytic 179

hemophilic bacillus. Arch. Int. Med 32:911-926. 180

15. Mukundan, D., Z. Ecevit, M. Patel, C. F. Marrs, and J. R. Gilsdorf. 2007. 181

Pharyngeal colonization dynamics of Haemophilus influenzae and Haemophilus 182

haemolyticus in healthy adult carriers. Journal of clinical microbiology 45:3207-3217. 183

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

9

16. Murphy, T. F., A. L. Brauer, S. Sethi, M. Kilian, X. Cai, and A. J. Lesse. 2007. 184

Haemophilus haemolyticus: a human respiratory tract commensal to be distinguished 185

from Haemophilus influenzae. J Infect Dis 195:81-89. 186

17. Norskov-Lauritsen, N. 2009. Detection of cryptic genospecies misidentified as 187

Haemophilus influenzae in routine clinical samples by assessment of marker genes 188

fucK, hap, and sodC. Journal of clinical microbiology 47:2590-2592. 189

18. Norskov-Lauritsen, N., M. D. Overballe, and M. Kilian. 2009. Delineation of the 190

species Haemophilus influenzae by phenotype, multilocus sequence phylogeny, and 191

detection of marker genes. Journal of bacteriology 191:822-831. 192

19. Pittman, M. 1953. A classification of the hemolytic bacteria of the genus 193

Haemophilus: Haemophilus haemolyticus Bergey et al. and Haemophilus 194

parahaemolyticus nov spec. Journal of bacteriology 65:750-751. 195

20. Sacchi, C. T., D. Alber, P. Dull, E. A. Mothershed, A. M. Whitney, G. A. Barnett, 196

T. Popovic, and L. W. Mayer. 2005. High level of sequence diversity in the 16S 197

rRNA genes of Haemophilus influenzae isolates is useful for molecular subtyping. 198

Journal of clinical microbiology 43:3734-3742. 199

21. Shapiro, E. D., and J. I. Ward. 1991. The epidemiology and prevention of disease 200

caused by Haemophilus influenzae type b. Epidemiologic reviews 13:113-142. 201

22. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular 202

Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596-203

1599. 204

23. Theodore, M. J., R. D. Anderson, X. Wang, L. S. Katz, J. T. Vuong, M. E. Bell, B. 205

A. Juni, S. A. Lowther, R. Lynfield, J. R. Macneil, and L. W. Mayer. 2012. 206

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

10

Evaluation of new biomarker genes for differentiating Haemophilus influenzae from 207

Haemophilus haemolyticus. Journal of clinical microbiology. 208

24. Wang, X., R. Mair, C. Hatcher, M. J. Theodore, K. Edmond, H. M. Wu, B. H. 209

Harcourt, G. Carvalho Mda, F. Pimenta, P. Nymadawa, D. Altantsetseg, M. 210

Kirsch, S. W. Satola, A. Cohn, N. E. Messonnier, and L. W. Mayer. 2011. Detection 211

of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR 212

assay to detect Haemophilus influenzae. International journal of medical microbiology : 213

IJMM 301:303-309. 214

215

216

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

11

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.

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

12

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

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from

13

264

265

266

267

on Decem

ber 23, 2018 by guesthttp://jcm

.asm.org/

Dow

nloaded from