Title: Comamonas kerstersii bacteremia in a patient with diverticulosis 1

2

Onya Opota1, Barbara Ney2, Giorgio Zanetti2, 3, Katia Jaton1, Gilbert Greub1, 2 and Guy Prod’hom1 3

Institute of Microbiology1, Infectious Diseases Service2, Hospital Preventive Medicine and Infectious 4

Diseases Service3, University Hospital of Lausanne, Lausanne, Switzerland 5

6

Corresponding author: 7

Gilbert Greub, 8

Institute of Microbiology, 9

University Hospital of Lausanne, 10

Bugnon 46, 1010 Lausanne, 11

Switzerland. 12

Phone +41 (0)21 314 4979 13

Fax +41 (0)21 314 4060 14

E-mail : Gilbert.Greub@chuv.ch 15

16

Keywords: Comamonas kerstersii, bacteremia, intra-abdominal infection, Delftia acidovorans, MALDI-17

TOF. 18

Abstract: 40 words 19

Text: 1774 words 20

21

JCM Accepts, published online ahead of print on 26 December 2013J. Clin. Microbiol. doi:10.1128/JCM.02942-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 22

We report for the first time a case of bacteremia caused by Comamonas kerstersii in a 65-year-old patient 23

with sign of diverticulosis. In addition, we review the isolation of Comamonas sp. and related organisms 24

in our hospital over 25 years. 25

26

CASE REPORT 27

Comamonas kerstersii is a non-fermenting β-Proteobacteria described in 2003 that has long been 28

considered as non-pathogenic (21). This organism has been recently associated with intra-abdominal 29

infection consecutive to perforation of the digestive tract (1). Herein, we describe a case of polymicrobial 30

bacteremia due to Comamonas kerstersii and Bacteroides fragilis in a 65-year-old diabetic man that was 31

admitted to the emergency department of the hospital due to sudden onset of fever and chills. The patient 32

reported episodes of vomiting and diarrhea and mentioned that he drank water from a small river. Stool 33

cultures performed after the beginning of the antibiotic treatment did not disclose neither Salmonella, 34

Shigella, Aeromonas, Campylobacter species nor Comamonas kerstersii. The detection of Clostridium 35

difficile toxin A and B and glutamate dehydrogenase antigen was also negative using the commercial 36

Clostridium difficile detection kit from Techlab®. Blood cultures (two pairs of bottles) were drawn from 37

a peripheral vein and the patient was discharged under treatment with oral ciprofloxacin for a 38

gastroenteritis of unknown origin. The Blood cultures were processed into a BACTEC FX automated 39

blood culture system (Becton Dickinson, Sparks, MD). A first aerobic blood culture bottle became 40

positive sixteen hours and eight minutes after sampling and the Gram staining revealed the presence of 41

long filamentous Gram-negative bacilli (Figure 1). The bacterial identification by matrix-assisted laser 42

desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), Bruker Daltonics GmbH, 43

Leipzig, Germany ) was performed the same day using a protocol that we recently developed based on the 44

analyses of a bacterial pellet preparation from the blood bottles (6, 16, 17). The strain was identified as 45

Comamonas kerstersii, a Gram-negative non-fermentative bacterium, and prompted the hospitalization of 46

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the patient. The patient was afebrile at that time, but palpation of the left lower abdominal quadrant was 47

painful. An abdominal CT scan revealed diverticulosis without evidence of diverticulitis. Consecutively, 48

the anaerobic blood bottles from the same pair became positive 24 hours and two minutes after sampling 49

for Bacteroides fragilis. We monitored the following MIC (mg/L, using the E-test method, BioMérieux, 50

Lyon, France) for the Comamonas kerstersii strain: ceftazidime, 0.75; meropenem, 0.004; imipenem, 51

0.06; minocycline, 0.38; levofloxacin, 4; co-trimoxazole, >32; ciprofloxacin, 32. For the Bacteroides 52

fragilis strain, the MIC were: amoxicillin-clavulanate, 1.5; piperacillin-tazobac, 6; imipenem, 0.12; 53

meropenem, 0.025; metronidazole, 0.25; clindamycin, 16; ciprofloxacin, 32. A treatment of imipenem-54

cilastatine was given for 10 days and the patient recovered. The final diagnosis was a mixed bacteremia 55

with Comamonas kerstersii and Bacteroides fragilis in the setting of a diverticulosis. 56

57

Comamonads are Gram negative, non-fermentative bacteria, oxydase- and catalase-positive, largely 58

motile due to the presence of polar flagella. The Comamonas genus originally contained Comamonas 59

terrigena, Comamonas testosteroni (previously Pseudomonas testosteroni) and Comamonas acidovorans 60

(previously Pseudomonas acidovorans) (7). It now contains seventeen species while Comamonas 61

acidovorans has been separated from the Comamonas genus on the basis of 16S rRNA and is now known 62

as Delftia acidovorans (22). In 2003, Shigematsu and colleagues have described a new species Delftia 63

tsuruhatensis that is known to be able to cause bacteremia (15, 18, 20). Biochemical analysis often 64

misclassifies these two Delftia species. Delftia tsuruhatensis being absent from our MALDI-TOF 65

database, we can not exclude misclassifications. For this reason Delftia acidovorans refers to Delftia 66

acidovorans sensu lato, which includes Delftia tsuruhatensis. Although ubiquitously distributed in the 67

environment (soil and water), Comamonas and Delftia sp. are rarely associated with infections in humans. 68

However, several publications have incriminated Comamonas testosteroni and Delftia acidovorans in 69

particular in human diseases, including severe invasive infections such as bacteremia and meningitis (2, 4, 70

5, 8, 11, 12, 14, 22) . 71

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Comamonas kerstersii, described in 2003 (21), has recently been reported as an agent of intra-abdominal 72

infection by Almuzara and colleagues (1). The present case is the first report of Comamonas kerstersii 73

bacteremia. We initially performed the identification of the strain at the species level directly from the 74

positive blood bottle using MALDI-TOF (6, 16, 17). The MALDI-TOF spectral score for Comamonas 75

kerstersii was 2.176 according to the manufacturer scoring system. Since no other species reached a score 76

above 2.0, this identification was considered as valid at the species level (3). Subsequently, it was 77

recovered both from the blood agar plate (with a spectral score of 2.26), on which the growth was 78

maximal and from the “chocolate” agar plate that is supplemented with NAD (factor V) and hemin (factor 79

X). The reliability of the MALDI-TOF to discriminate between Comamonas species is shown by the fact 80

that Comamonas aquatica, the closest species also present in the MALDI-TOF database gave a score of 81

1.542. The data-base that was used contains five Comamonas species (Comamonas kerstersii, 82

Comamonas testosteroni, Comamonas aquatica, Comamonas terrigena and Comamonas nitrativorans) 83

and five strains of Delftia acidovorans. The recently identified species Delftia tsuruhatensis is absent 84

from this database (18) and we can not excluded that part of the Delftia acidovorans identified in our 85

laboratory using the MALDI-TOF could be Delftia tsuruhatensis. In the absence of MALDI-TOF, 86

numerous biochemical tests (at least 11) are necessary to discriminate Comamonas species (1, 21); such 87

identification requires isolation on agar plate and can not be performed the same day of positivity directly 88

from the blood culture. Because of the excellent identification obtained with the MALDI-TOF we rather 89

proceeded to the 16S sequencing. Using primers described by (24), we obtained a 780 bp partial 16S 90

rRNA sequence. The BLAST of this sequence on the NCBI website, revealed 99% of identities (754/755 91

nucleotides, with no gaps) with the Comamonas kerstersii sequence (accession number AJ430348.1) 92

submitted by Wauters and colleagues (21). The divergence corresponds to a polymorphism reported by 93

the authors at position 298 (21). This method is limited by the low number of Comamonas kerstersii 16S 94

rRNA sequences. Nevertheless, the first sequence identified as Comamonas aquatica, (accession number 95

FJ493173) displays 98% of identities (749/763, with 2 gaps).We also proceeded to the amplification and 96

sequencing of the 16S rRNA ribosomal gene in order to confirm the MALDI-TOF identification of this 97

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strain (9). The analysis of the sequences using the BLAST V2.0 software 98

(http://www.ncbi.nlm.nih.gov/BLAST/) showed 100% of identity with the sequences corresponding to 99

the 16S RNA ribosomal gene of Comamonas kerstersii strain LMG 5323 (21). From both the blood bottle 100

and the agar plates, the strain appeared as an extremely long Gram-negative filamentous bacillus which is 101

a very unusual phenotype for bacteria of this genus (Figure 1). The Comamonas and Delftia strains 102

previously isolated in our hospital are Gram-negative short bacilli or rods (Figure 1), which is the 103

morphology described for these organisms (21, 22). 104

Translocation from the digestive tract seems to be a predominant cause of infections by Delftia and 105

Comamonas species. Recently, Hagiya and colleagues reported a Delftia acidovorans bacteremia in a 46-106

year-old woman caused by translocation of the bacteria consecutive to pesticide poisoning (11). A 107

bacteremia caused by Comamonas testosteroni was previously reported, in a 22-year-old man with 108

perforated appendix (10). In the four cases reported by Almuzara and colleagues, the Comamonas 109

kerstersii strains were isolated from intra-abdominal collections (1). We previously identified another 110

Comamonas kerstersii strain in an intra-peritoneal collection of an 11-year-old child with a perforated 111

appendix (table 1 and figure 1). Herein, the digestive origin of the Comamonas kerstersii strain is 112

supported by the fact that: i) the patient reported abdominal pain, vomiting and diarrhea, ii) the CT scan 113

revealed evidence of diverticulosis, and iii) the enteric bacteria Bacteroides fragilis was isolated from the 114

blood culture. Bacteroides fragilis is a gut commensal of humans that can cause severe disease such as 115

bacteremia and abscesses due to production of several virulence factors such as capsule, endotoxins and 116

enterotoxins (23). Comamonas kerstersii infection could originate from the water that the patient drank in 117

the countryside. 118

Comamonas species have been rarely associated with infection in humans despite their ubiquitous 119

distribution in the environment possibly due to the difficulty to accurately distinguish Comamonas 120

species from Pseudomonas species in the pre-MALDI-TOF era (1). Alternatively, Comamonads could 121

have been under-recognized due to their common occurrence in the setting of a polymicrobial infection. 122

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In our 1027-bed tertiary care university hospital, thirty-two Comamonas sp. strains and thirty-eight 123

Delftia acidovorans strains where isolated from 1997 to 2013. They were primarily isolated from 124

respiratory samples (33%), urogenital samples (23%) and digestive samples (21%); bacteremia 125

represented 5% (three patients) of all cases (Table 1). The 3 bacteremias had distinct clinical features but 126

were all poly-microbial (table 2). The first bacteremia case was due to Comamonas testosteroni in 127

association with Streptococcus parasanguis and Ralstonia pickettii in a 33-year old man. A second case 128

involved Delftia acidovorans in association with Streptococcus agalactiae in blood cultures from a 61-129

year-old man. The last case is the present Comamonas kerstersii and Bacteroides fragilis co-infection. 130

Like Delftia acidovorans, Comamonas testosteroni is the Comamonas species predominantly associated 131

with bacteremia (table 1) (8, 10, 11, 14, 22). Translocation from the digestive tract and catheters are the 132

predominant source of infection (10, 11, 13, 14). Immunocompromised children or patients with 133

compromised immune systems such as patients with acquired immune deficiency syndrome (AIDS) or 134

patients with a cancer or treated with aplastic chemotherapies, appear to be particularly at risk to develop 135

Comamonas sp. or Delftia acidovorans bacteremia (12, 13). Interestingly, Khan and colleagues reported a 136

fatal outcome in an 4-year old immuno-competent child presenting a Delftia acidovorans bacteremia 137

(12). The patient presented herein did not display any sign of immunodeficiency suggesting that such 138

bacteremia may also occur in the absence of immunosuppression. The likely high inoculum in the water 139

that was drunk and the diabetic status of the patient are two significant co-factors that may explain the 140

occurrence of a bacteremia in the setting of a gastro-intestinal infection. Similarly, a Comamonas species 141

bacteremia has been associated with exposure to possibly contaminated water of a fish tank (19). 142

Comamonas kerstersii has long been considered as non-pathogenic based on the lack of 143

association with severe infections. This could in part be explained by the recent description of this species 144

and to the difficulties to accurately identify it. This first report of poly-microbial bacteremia involving 145

Comamonas kerstersii reveals that this organism can be involved in severe diseases. Comamonas 146

kerstersii pathogenicity could be due to the versatility of this organism, which makes it able to grow in 147

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various conditions. This report highlights the usefulness of MALDI-TOF in rapidly and accurately 148

identifying non-fermentative Gram negative bacteria that were difficult to identify in the pre-MALDI-149

TOF era. This could help at redefining the epidemiology and clinical syndromes due to these organisms. 150

ACKNOWLEDGEMENTS 151

We are grateful to all the technicians of the Diagnostic Microbiology Laboratory of the Lausanne 152

Hospital for their technical contribution. In particular, we thank Maria Senra-Ortiz and Christian 153

Durussel. 154

REFERENCES 155

1. Almuzara, M. N., R. Cittadini, C. Vera Ocampo, R. Bakai, G. Traglia, M. S. Ramirez, M. 156 del Castillo, and C. A. Vay. 2013. Intra-abdominal infections due to Comamonas kerstersii. J 157 Clin Microbiol 51:1998-2000. 158

2. Arda, B., S. Aydemir, T. Yamazhan, A. Hassan, A. Tunger, and D. Serter. 2003. Comamonas 159 testosteroni meningitis in a patient with recurrent cholesteatoma. APMIS 111:474-6. 160

3. Bizzini, A., C. Durussel, J. Bille, G. Greub, and G. Prod'hom. 2010. Performance of matrix-161 assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial 162 strains routinely isolated in a clinical microbiology laboratory. J Clin Microbiol 48:1549-54. 163

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5. Cooper, G. R., E. D. Staples, K. A. Iczkowski, and C. J. Clancy. 2005. Comamonas 167 (Pseudomonas) testosteroni endocarditis. Cardiovasc Pathol 14:145-9. 168

6. Croxatto, A., G. Prod'hom, and G. Greub. Applications of MALDI-TOF mass spectrometry in 169 clinical diagnostic microbiology. FEMS Microbiol Rev 36:380-407. 170

7. De Vos, P., K. Kersters, E. Falsen, B. Pot, M. Gillis, P. Segers, and J. De Ley. 1985. 171 Comamonas Davis and Park 1962 gen. nov., nom. rev. emend., and Comamonas terrigena Hugh 172 1962 sp. nov., Int. J. Syst. Bacteriol. 35.:443– 453. 173

8. Ender, P. T., D. P. Dooley, and R. H. Moore. 1996. Vascular catheter-related Comamonas 174 acidovorans bacteremia managed with preservation of the catheter. Pediatr Infect Dis J 15:918-175 20. 176

9. Goldenberger, D., T. Schmidheini, and M. Altwegg. 1997. Detection of Bartonella henselae 177 and Bartonella quintana by a simple and rapid procedure using broad-range PCR amplification 178 and direct single-strand sequencing of part of the 16S rRNA gene. Clin Microbiol Infect 3:240-179 245. 180

10. Gul, M., P. Ciragil, E. Bulbuloglu, M. Aral, S. Alkis, and F. Ezberci. 2007. Comamonas 181 testosteroni bacteremia in a patient with perforated acute appendicitis. Short communication. 182 Acta Microbiol Immunol Hung 54:317-21. 183

11. Hagiya, H., T. Murase, J. Sugiyama, Y. Kuroe, H. Nojima, H. Naito, S. Hagioka, and N. 184 Morimoto. 2013. Delftia acidovorans bacteremia caused by bacterial translocation after 185 organophosphorus poisoning in an immunocompetent adult patient. J Infect Chemother 19:338-186 41. 187

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12. Khan, S., S. Sistla, R. Dhodapkar, and S. C. Parija. 2012. Fatal Delftia acidovorans infection 188 in an immunocompetent patient with empyema. Asian Pac J Trop Biomed 2:923-4. 189

13. Le Moal, G., M. Paccalin, J. P. Breux, F. Roblot, P. Roblot, and B. Becq-Giraudon. 2001. 190 Central venous catheter-related infection due to Comamonas testosteroni in a woman with breast 191 cancer. Scand J Infect Dis 33:627-8. 192

14. Nseir, W., J. Khateeb, M. Awawdeh, and M. Ghali. 2011. Catheter-related bacteremia caused 193 by Comamonas testosteroni in a hemodialysis patient. Hemodial Int 15:293-6. 194

15. Preiswerk, B., S. Ullrich, R. Speich, G. V. Bloemberg, and M. Hombach. 2011. Human 195 infection with Delftia tsuruhatensis isolated from a central venous catheter. J Med Microbiol 196 60:246-8. 197

16. Prod'hom, G., A. Bizzini, C. Durussel, J. Bille, and G. Greub. 2010. Matrix-assisted laser 198 desorption ionization-time of flight mass spectrometry for direct bacterial identification from 199 positive blood culture pellets. J Clin Microbiol 48:1481-3. 200

17. Prod'hom, G., C. Durussel, and G. Greub. 2013. A simple blood-culture bacterial pellet 201 preparation for faster accurate direct bacterial identification and antibiotic susceptibility testing 202 with the VITEK 2 system. J Med Microbiol 62:773-7. 203

18. Shigematsu, T., K. Yumihara, Y. Ueda, M. Numaguchi, S. Morimura, and K. Kida. 2003. 204 Delftia tsuruhatensis sp. nov., a terephthalate-assimilating bacterium isolated from activated 205 sludge. Int J Syst Evol Microbiol 53:1479-83. 206

19. Smith, M. D., and J. D. Gradon. 2003. Bacteremia due to Comamonas species possibly 207 associated with exposure to tropical fish. South Med J 96:815-7. 208

20. Tabak, O., B. Mete, S. Aydin, N. M. Mandel, B. Otlu, R. Ozaras, and F. Tabak. 2013. Port-209 related Delftia tsuruhatensis bacteremia in a patient with breast cancer. New Microbiol 36:199-210 201. 211

21. Wauters, G., T. De Baere, A. Willems, E. Falsen, and M. Vaneechoutte. 2003. Description of 212 Comamonas aquatica comb. nov. and Comamonas kerstersii sp. nov. for two subgroups of 213 Comamonas terrigena and emended description of Comamonas terrigena . Int J Syst Evol 214 Microbiol 53:859-62. 215

22. Wen, A., M. Fegan, C. Hayward, S. Chakraborty, and L. I. Sly. 1999. Phylogenetic 216 relationships among members of the Comamonadaceae, and description of Delftia acidovorans 217 (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov. Int J Syst Bacteriol 49 218 Pt 2:567-76. 219

23. Wexler, H. M. 2007. Bacteroides: the good, the bad, and the nitty-gritty. Clin Microbiol Rev 220 20:593-621. 221

24. Zucol, F., R. A. Ammann, C. Berger, C. Aebi, M. Altwegg, F. K. Niggli, and D. Nadal. 2006. 222 Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by 223 sequencing for species identification. J Clin Microbiol 44:2750-9. 224

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Figure legend: 229

Figure 1: Gram staining of Comamonas kerstersii, Comamonas testosteroni and Delftia acidovorans 230

strains isolated in clinical samples from Lausanne University Hospital. (A) The Comamonas 231

kerstersii strain of the present case report directly from the blood-cultures bottle or B after culture on 232

blood agar medium. The strain displays long filaments when compared to the other strains that appear as 233

Gram-negative short bacilli or rods. C) A Comamonas kerstersii strain identified in an intra-peritoneal 234

collection of an 11-year-old child with a perforated appendix. D) Comamonas testosteroni involved in a 235

bacteremia in a 33-year- old man. E) Delftia acidovorans identified in blood cultures from a 61-year-old 236

man 237

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Table 1: Comamonas sp. and Delfia acidovorans isolated from clinical samples in the Lausanne 239

University Hospital from 1997 to 2013. 240

241

Respiratory

and ENTa

Urogenitalb Intra-

abdominalc

Skin Blood culture

Surgical wound Others

d Total

patient (samples)

% of patient

Deltia acidovorans

19 (20)

12 (13)

- 5 1 (13)

- 1 (4)

38 (55)

54.3

Comamonas testosteroni

2 (4)

3 8 1 (4)

1 4 (5)

1 20 (26)

28.57

Comamonas kerstersii

- - 1 - 1 - - 2 2.86

Comamonas aquatica

- 1 (4)

- - - - - 1 (4)

1.43

Comamonas specis

(e) 2 -

(2) 6

(8) 1 - - -

(1) 9

(14) 12.86

Number of patients (number of samples)

23 (26)

16 (22)

15 (17)

7 (10)

3 (15)

4 (5)

2 (6)

70 (101)

100

Percentage of patients (percentage of samples)

32.86 (25.74)

22.86 (21,78)

21.43(16,83)

10(9.9)

4.29(14.85)

5.71 (4.95)

2.85 (5.94)

100

aEar nose and thoat

bUrine, vaginal swab and placenta

cascitic fluid, peritoneal fluid, penrose liquid, kehr drain

dstools (1), bone fragment (1), orifice smear (1)

eno identification at the species level

Number represent patients, brackets represent the number of samples 242

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Table 2: Summary of clinical and microbiological bacteremia due to Comamonas testosteroni, 243

Comamonas kerstersii and Delftia acidovorans and of a case of intra-abdominal collection of 244

Comamonas kerstersii. 245

246 247

Age (year),

sex

Site of infection

Clinical presentation

Undelying diseases

Predisposing factors

Pathogens Treatment

61, male Blood Fever Lymphoma Chimiotherapy,

aggranulocytosis

Delftia acidovorans, Streptococcus

agalactiae cefepime followed by

imipenem and ciprofloxacine

33, male Blood

High fever, hypotension,

infected phlebitis on

the arm due to cocaine injection

Chronique hepatitis C,

chronic alcoholism, drug addict

No predisposing factor

Comamonas testosteroni,

Streptococcus parasanguis, Ralstonia

pickettii

Cefepime and vancomycin followed

by moxifloxacin

65, male* Blood

Fever, chills, vomiting and

diarrhea Diabetic No predisposing

factor Comamonas kerstersii,

Bacteroides fragilis ciprofloxacin,

imipenem

12, male Peritoneal fluid

Abdominal pain

No underlying disease Appendicitis

Comamonas kerstersii, Escherichia coli, Streptococcus spp

(group anginosus/milleri)

co-amoxicillin, metronidazole and

amikacin followed by co-amoxicillin alone

*present case

248

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