persistent blood stream infection with kocuria rhizophila
TRANSCRIPT
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Persistent blood stream infection with Kocuria rhizophila related to a 1
damaged central catheter 2
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Didier Moissenet1*, Karsten Becker2, Audrey Mérens3, Agnès Ferroni4, Béatrice Dubern5, 5
and Hoang Vu-Thien1 6
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1) Assistance Publique-Hôpitaux de Paris, Hôpital Armand-Trousseau, Service de 11
Microbiologie, Faculté de Médecine, Université Pierre et Marie Curie-Paris 6, Paris, France 12
2) University Hospital of Münster, Institute of Medical Microbiology, Münster, Germany 13
3) Hôpital d’Instruction des Armées Bégin, Service de Biologie Médicale, Saint Mandé, 14
France 15
4) Assistance Publique-Hôpitaux de Paris, Hôpital Necker, Service de Microbiologie, Faculté 16
de Médecine, Université René Descartes-Paris 5, Paris, France 17
5) Assistance Publique-Hôpitaux de Paris, Hôpital Armand-Trousseau, Service de Gastro-18
Entérologie-Nutrition, Faculté de Médecine, Université Pierre et Marie Curie-Paris 6, Paris, 19
France 20
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*Corresponding author. Mailing address: Service de Microbiologie, Hôpital Armand-28
Trousseau, 26 av. du Dr A. Netter, 75571 Paris Cedex 12, France 29
Tél: 33 1 44 73 61 43 30
Fax: 33 1 44 73 53 29 31
Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.06038-11 JCM Accepts, published online ahead of print on 18 January 2012
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A case of persistent blood stream infection with Kocuria rhizophila related to a 33
damaged central venous catheter in a 3-year-old girl with Hirschprung’s disease is 34
reported. The strain was identified as K. rhizophila by 16S rRNA gene sequencing and 35
matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Arbitrarily 36
primed PCR analysis showed a clonal strain. The repeated septic episodes were resolved 37
with the catheter repair. 38
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CASE REPORT 40
The patient was a 3-year-old girl with total colonic form of Hirschprung’s disease. On 41
day 2 of life (27th April 2006), an emergency surgery for small intestine occlusion due to 42
atresia was performed with removal of 31 cm of small intestine, followed by terminal 43
ileostomy and colostomy. A total parenteral nutrition was then needed. On 25th August 2006, 44
a subcutaneous implantable vascular-access port (Cook Spectrum Central Venous Catheter, 45
Cook Ireland LTD.) was placed for home parenteral nutrition after an accidental removal of 46
the Nutricath (Vygon, France) catheter. 47
Four months later (December 2006), the first septic episode was observed with seven 48
positive blood samples drawn through the catheter but also from a peripheral vein. The fever 49
resolved promptly after the initiation of antimicrobial therapy combining vancomycin (40 50
mg/kg/day, 10 days) and gentamicin (3 mg/kg/day, 2 days). Blood isolates were firstly 51
identified as Micrococcus sp. with routine biochemical galleries, and subsequently as Kocuria 52
rhizophila with molecular tools. Afterwards, seven other septic episodes with K. rhizophila 53
(two in 2007, four in 2008, and one in 2009) were observed and resolved with the same 54
antimicrobial therapy. Since it was assumed that colonization of the catheter was the cause of 55
sepsis, ethanol locks (instillation of 70% ethanol into the catheter lumen during 12 hours, 56
withdrawn, isotonic sodium chloride flush) were made in the catheter during the four last 57
episodes associated with systemic antibiotics (17). At the time of the last septic event in 2009, 58
hole in catheter was detected and repaired using specific repairing kit. After April 2009, no 59
novel septic episode was observed. 60
During the 3-year period (2007-2009), using the BacT/ALERT 3D system 61
(bioMérieux, Marcy-l’Etoile, France), a total of 22 positive blood cultures were obtained, 62
with 21 samples drawn from the catheter and once from a peripheral vein. All the cultures 63
yielded gram-positive cocci occurring in pairs, tetrads and clusters that were preliminarily 64
identified as Micrococcus species with basic characteristics. The colonies grew under aerobic 65
conditions, and appeared smooth and circular with a yellow lemon tinge or creamy on blood 66
agar. The isolates were catalase positive, oxidase negative, susceptible to bacitracin and 67
resistant to furazolidone. Only a few positive reactions were found with the ID 32 Staph 68
gallery (bioMérieux), yielding Staphylococcus auricularis with weak probability of 57%. In 69
2006-2007, the clinical strain was considered as Micrococcus without other investigation in 70
the identification process. In 2008, we used the Vitek 2 ID-GPC card (bioMérieux) that 71
yielded Kocuria varians with an apparent ‘excellent’ confidence score (98%). It showed only 72
a single discordant test (urea) since it appeared negative for this species whereas it should be 73
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positive in 87% of K. varians isolates. In order to clarify this discordant test and confirm K. 74
varians, we used 16S rRNA gene analysis. DNA extraction and 16S rRNA gene sequence 75
analysis were performed as previously described (13). The procedure was performed on 11 76
clinical isolates: four recovered in 2006, six in 2008 and one in 2009. Surprisingly, all the 77
sequences obtained showed a complete identity to those of K. rhizophila deposited in the 78
GenBank nucleotide database. The 16S rRNA gene sequence of K. rhizophila isolate has been 79
submitted to the GenBank nucleotide database under GenBank accession number JQ272742. 80
This sequence showed a similarity of 98% (457/466) with the 16S rRNA gene of the strain K. 81
rhizophila DC22201 (GenBank accession no. NC010617), as well as ten other K. rhizophila 82
strains of which Kovacs’historic type strain K. rhizophila TA68T (GenBank accession no. 83
NR_026452, similarity of 99%), and only 93% with Micrococcus luteus NCTC 2665 84
(GenBank accession no. NC012803). Other Kocuria species showed a similarity of 451/462n 85
for Kocuria carniphila and 452/467n for Kocuria marina. Finally and recently, we have used 86
matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-87
MS) as previously described (6), confirming the strain as K. rhizophila. The spectral profile of 88
four clinical isolates were identical to the spectral profile of the K. rhizophila type strain (Fig. 89
1) and quite distinct from the profiles of Kocuria kristinae, Kocuria palustris, Kocuria rosea 90
and Kocuria varians, all present in Andromas® database. With the disk diffusion method, all 91
the isolates were resistant to ciprofloxacin, intermediate to erythromycin, and susceptible to 92
penicillin, gentamicin, amikacin, tobramycin, tetracycline, vancomycin and teicoplanin, 93
according to EUCAST breakpoints determined for Staphylococcus spp since there are not 94
breakpoints available for Kocuria spp. With broth microdilution, MICs were 0.03 mg/l for 95
penicillin, 0.5 mg/l for gentamicin, 1 mg/l for amikacin, 2 mg/l for tobramycin, 8 mg/l for 96
ciprofloxacin, 4 mg/l for erythromycin, 0.125 mg/l for tetracycline, 0.5 mg/l for vancomycin 97
and 1 mg/l for teicoplanin. 98
Clinical isolates genotyping with the arbitrarily primed PCR technique needed 99
prolonged ramp times, as reported by Ellinghaus et al. (8). It was performed on 9 100
representative isolates of the eight septic episodes, and showed the same pattern, representing 101
a clonal K. rhizophila strain recovered for 3 years (Fig. 2). 102
In 1995, the genus Micrococcus was dissected into five genera (Kocuria, 103
Nesterenkonia, Kytococcus, Dermacoccus and Micrococcus) (23) with description of three 104
Kocuria species (K. rosea, K. varians and K. kristinae). Here, the trivial terms “micrococci” 105
as well as “micrococcal” were used in quotation marks to indicate members of these genera. 106
The genus Kocuria belongs to the family of Micrococcaceae, which is part of the order of 107
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Actinomycetales, class Actinobacteria. In 1999, K. rhizophila was described with K. palustris 108
by Kovacs et al. (10). It was isolated from the rhizoplane of the narrow-leaved cattail (Typha 109
angustifolia) inhabiting a floating mat on a creek of the Hungarian part of the Danube River. 110
In 2003, the strain ATCC 9341, originally deposited as Sarcina lutea and later redesignated 111
Micrococcus luteus, was reclassified as K. rhizophila (25). From 2004, eleven other Kocuria 112
species were described (K. marina sp. nov. (9), K. carniphila sp. nov. (28), K. aegyptia sp. 113
nov. (14) K. himachalensis sp. nov. (16), K. flava sp. nov. and K. turfanensis sp. nov. (32), K. 114
halotolerans sp. nov. (26), K. gwangalliensis sp. nov. (22), K. koreensis sp. nov. (20), K. 115
atrinae sp. nov. (19) and K. salsicia sp. nov. (31)). K. rhizophila is also important in industrial 116
applications and is commonly used as a standard quality control strain for antimicrobial 117
susceptibility testing. In 2008, the complete genome sequence of K. rhizophila was 118
determined by Takarada et al. (24). 119
“Micrococcal” species were found in dust, soil, water, food and on skin and mucosa of 120
humans and animals. Members of these species group have also been found to cause 121
infections such as meningitis, endocarditis, pneumonia, particularly in immunocompromised 122
patients and infections related to implanted or inserted devices (18, 21, 30). Among the 123
recently established genus Kocuria, documented human cases of infections are limited. The 124
type species K. rosea has been reported to cause catheter-related bacteremia (2). Another 125
member of the genus, K. kristinae has been also reported to cause also a catheter-related 126
bacteremia in patients with ovarian cancer (3) or acute cholecystitis (15). In 2009, two cases 127
of peritonitis caused by K. marina were reported by Lee et al. (12). More recently in 2010, 128
Lai et al. (11) reported catheter-related bacteremia and infective endocarditis caused by 129
Kocuria sp., whereas Tsai et al. (27) reported a K. varians infection associated with brain 130
abscess. 131
While K. rhizophila has been isolated from food such as cheese (7) or chicken meat 132
(1), to our knowledge, our case is the second report of K. rhizophila human infection after the 133
first one described by Becker et al. in 2008 (4). While the source of our 3-years persistent K. 134
rhizophila strain was unclear, it is possible that it was a part of resident skin flora of the 135
patient, colonizing the intravascular device on several occasions. The long-term intravascular 136
device (3 years) probably provided a niche for the persistent K. rhizophila strain, recurring 137
through the hole in the device. Repair of the damaged device seemed to prevent the 138
occurrence of any novel septic episode until now. Obviously, the strict application of aseptic 139
protocol concerning central venous catheters management is now frequently reminded to the 140
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whole ward staff. Currently, the child is in good health with always liquid stools. Parenteral 141
nutrition could be reduced to 5 days per week. 142
The ID 32 Staph gallery did not allow a reliable identification of K. rhizophila since 143
the database of this commercially available diagnostic kit include only a limited number of 144
“micrococcal” species, not covering the recently described species and not reflecting the 145
taxonomy established by Stackebrandt et al. (23). The Vitek 2 ID-GP card ambiguously 146
identified several Kocuria species (K. varians, but also K. kristinae or K. rosea) except K. 147
rhizophila. Moreover, misidentification of coagulase negative staphylococci as Kocuria using 148
standard biochemical analysis by the Vitek 2 system is not uncommon, due to phenotypic 149
variability (5). In contrast, the use of 16S rRNA gene analysis or MALDI-TOF-MS was 150
adequate to obtain an accurate identification of K. rhizophila. 151
Few data are currently available about antimicrobial susceptibility of Kocuria sp. or 152
other “micrococci” and, moreover, no generally accepted therapeutic regimen for severe 153
infections has yet been defined. In 1995, von Eiff et al. (29) determined minimal inhibitory 154
concentrations (MICs) of several drugs on 188 “micrococcal” strains: MICs90 (mg/l) of 155
rifampin, penicillin, imipenem, ampicillin, clindamycin, cefotaxime, vancomycin/teicoplanin, 156
gentamicin were respectively ≤0.031, 0.125, 0.125, 0.25, 0.25, 1, 1/1 and 1, whereas MICs90 157
of amikacin, erythromycin, fosfomycin and fusidic acid were >2 mg/l. In our case, the strain 158
was susceptible to vancomycin and gentamicin, and the combination of these two drugs 159
always allowed sterilization of blood cultures. 160
In conclusion, if an organism resembling “micrococci” is repeatedly isolated from 161
blood cultures, it is important to use other means than the routine biochemical systems, such 162
as 16S rRNA gene sequencing or MALDI-TOF-MS, to obtain an accurate species 163
identification. It is also recommended to verify carefully the integrity of long-term 164
intravascular devices and repair an eventual damage to salvage central lines from removal. 165
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FIG. 1. MALDI-TOF-MS spectral profiles. First to 4th line: four clinical K. rhizophila 242 isolates from our patient. Fifth and last line: K. rhizophila type strain used in Andromas 243 database.244
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FIG. 2. Arbitrarily primed PCR patterns of K. rhizophila strains. M: DNA ladder. Lanes 1 247
(type strain DSM 11926) and 11 (K. rhizophila from another patient): unrelated strains. Lanes 248
2-3: two isolates from the first septic episode. Lane 4: isolate from the second episode. Lane 249
5: isolate from the third episode. Lane 6: isolate from the fourth episode. Lane 7: isolate from 250
the fifth episode. Lane 8: isolate from the sixth episode. Lane 9: isolate from the seventh 251
episode. Lane 10: isolates from the last septic episode. Lanes 2 to 10: similar patterns 252
indicating the same strain. Lane 12: negative control. 253
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