After a taxonomic revision of Micrococcus was made by Stackebrandt et al.  in 1995, this genus was separated into the genera, Mirococcus, Nesterenkonia, Kytococcus and Dermacocccus as well as the new genus Kocuria. Kocuria is a member of the Micrococcus family, which includes Kocuria rosea, Kocuria kristinae, Kocuria varians, Kocuria palustris, Kocuria rhizophila, Kocuria marina and Kocuria aegyptia [1–5]. In the limited number of reported cases, the clinical manifestations of Kocuria infection included central venous catheter-related bloodstream infection and acute cholecystitis [5–8]. However, Ben-Ami et al.  reported on the misidentification of coagulase-negative Staphylococcus, which often caused catheter related infection, as Kocuria species by the VITEK 2 system (bioMerieux, Marcy l’Etoile, France) and suggested that the precise identification of these rare microorganisms requires genomic sequencing, a method that is not available in most clinical microbiology laboratories. To better understand the epidemiology and the clinical characteristics of infections caused by these environmental pathogens, we describe five patients with positive blood cultures for Kocuria species.
From January 2006 to June 2009, patients who had a positive blood culture for Kocuria species in the National Taiwan University Hospital were included. Identification of the Kocuria genus was based on conventional biochemical tests and on results obtained using the Phoenix automated system PMIC/ID-30 (Becton Dickinson Diagnostic Systems, Sparks, MD, USA) and the VITEK 2 system (bioMérieux Inc., La Balme les Grottes, France). A partial sequence of the 16S rRNA gene (548 bp) was generated to confirm the identification of our isolates . A pair of universal primers (primers 27F and 1492R) were used . The sequences obtained were compared with published sequences in the GenBank database by using the blastn algorithm (http://www.ncbi.nlm.nih.gov/blast). The closest matches and GenBank accession numbers were obtained. MICs of the isolates were determined by the broth microdilution method using the Phoenix automated system PMIC/ID-30 (Becton Dickinson Diagnostic Systems).
Random amplified polymorphic DNA (RAPD) patterns of all isolates of Kocuria species generated by arbitrarily primed PCR were as described previously . Two arbitrary oligonucleotide primers: M13 (5′-GAGGGTGGCGGTTCT-3′) (Gibco BRL Products, Gaithersburg, MD, USA) and H12 (5′-ACGCGCATGT-3′) (Operon Technologies, Inc., Alameda, CA, USA) were used. The arbitrarily primed PCR analyses of these isolates were performed in duplicate. Interpretation of the RAPD patterns was performed as previously described .
A total of 21 blood isolates from five patients were identified as Kocuria species by conventional biochemical methods. Twenty isolates of K. kristinae, which were initially identified by the Phoenix automated system (confidence value 99%) and VITEK 2 system (probability of identification 99%), were confirmed by 16S rRNA sequencing [accession number of all isolates was EU379300.1, maximal identity, 99.8% (547/548)]. One isolate, initially identified as K. varians by the Phoenix automated system (confidence value 99%) and K. kristinae by the VITEK 2 system (probability of identification 96.71%), respectively, was finally confirmed to be K. marina by 16S rRNA gene sequencing [accession number FJ789550.1, maximal identity, 100% (548/548)]. The MIC ranges of the five Kocuria species to 20 antimicrobial agents are shown in Table 1.
Table 1. Susceptibilities of five nonduplicate isolates of Kocuria species to 20 antimicrobial agents using the Phoenix automated system PMIC/ID-30 (Becton Dickinson Diagnostic Systems)
|Agent||MIC range (mg/L)|
|Kocuria kristinae (n = 4)||Kocuria marina (n = 1)|
|Nitrofurantion||32 to >64||>64|
The clinical characteristics of the five patients with Kocuria bacteraemia are summarized in Table 2. In patient 3, the isolates probably reflected contamination since only one bottle (aerobic bottle) of two sets of blood cultures grew the organism and this patient did not receive antimicrobial therapy. Patient 1 had infective endocarditis and another three (patients 2, 4 and 5) patients had catheter-related bacteraemia. All infections were successfully managed and there was no infection-related mortality.
Table 2. Clinical characteristics of five patients with positive blood cultures for Kocuria species
|Patient number (year)||Age (years)/sex||Underlying diseases or condition||Kocuria species identified by Phenix/VITEK 2 (no. of isolates)||Kocuria species identified by 16S rRNA GenBank (accession number)||Source of infection||Antimicrobial therapy||Removal of the catheter||Outcome|
|1 (2006)||89/F||Ischaemic bowel disease status post operation, short bowel syndrome, TPN use, Port A Catheter||Kocuria kristinae/Kocuria kristinae (8)||Kocuria kristinae (EU379300.1)||Infective endocarditis||Vancomycin teicoplanin oxacillin||Yes||Improved|
|2 (2007)||37/F||Gastric cancer, Port A Catheter, TPN use||Kocuria kristinae/Kocuria kristinae (3)||Kocuria kristinae (EU379300.1)||Catheter- related bacteraemia||Piperacillin-tazobactam ciprofloxacin||Yes||Improved|
|3 (2007)||57/F||Lung cancer, Port A Catheter||Kocuria varians/Kocuria kristinae (1)||Kocuria marina (FJ789660.1)||Contamination||No||No||NA|
|4 (2009)||2/M||Congenital short bowel syndrome, hypogammaglobulemia, Port A Catheter, TPN use||Kocuria kristinae/Kocuria kristinae (7)||Kocuria kristinae (EU379300.1)||Catheter- related bacteraemia||Oxacillin + lock-therapy with vancomycin||Yes||Improved|
|5 (2009)||68/F||Gastric cancer, TPN use, Port A Catheter||Kocuria kristinae/Kocuria kristinae (2)||Kocuria kristinae (EU379300.1)||Catheter- related bacteraemia||Oxacillin||Yes||Improved|
RAPD pattern analysis showed that isolates from the four patients all had different RAPD patterns, indicating that they were genetically unrelated and that nosocomial transmission had not occurred. Second, multiple isolates from these four patients with persistent CRBSI had identical RAPD patterns, implying either incomplete treatment for an occult focus or persistent carriage of the same bacteria.
This report describes four patients with K. kristinae infection in immunocompromised patients, which suggests that Kocuria species are rare pathogens in immunocompromised adults and children. In addition, the present series includes the first reported case of endocarditis due to K. kristinae and expands the clinical spectrum of infections caused by this group of bacteria.
All four patients with K. kristinae infection in the present study had an implanted catheter and all of them had catheter removal prior to clearance of infection. This is consistent with a previously reported case in which persistent K. kristinae bacteraemia did not improve until catheter removal  and suggests that catheter removal should be recommended for the treatment of K. kristinate infection.
In this survey of 21 isolates identified as Kocuria species by conventional biochemical analysis, the findings with respect to 16S rRNA gene sequencing for 20 isolates of K. kristinae were consistent with the conventional test results. However, for another non-K. kristinae isolate, the results of 16S rRNA gene sequencing were different from the identification made by the Phoenix system and the VITEK 2 system. This finding suggests that accurate identification to the species level requires advanced molecular methods such as 16S rRNA gene sequencing, which are useful for the identification of clinically significant species Kocuria.
Data on the antibiotic susceptibility patterns of Kocuria species are very limited. Our results were in accordance with those obtained in a previous study . However, the four K. kristinae isolates had oxacillin MICs up to 4 mg/L, which were considered resistant to oxacillin if MIC interpretive breakpoints for S. aureus and S. lugdunensis (resistant, MICs ≥ 4 mg/L) or for other coagulase-negative staphylococci (resistant, MICs ≥ 0.5 mg/L) were applied . Despite the difficulty in drawing inferences about an optimal antibiotic treatment based on this limited experience, our data suggest that the clinical microbiology laboratory has the ability to identify these unusual bacteria to the species level because as a result of the different drug susceptibility patterns for the different Kocuria species.
In conclusion, Kocuria is an uncommon cause of infection in immunocompromised patients. The clinical manifestations are protean, and include catheter-related infection and infective endocarditis. Accurate identification with molecular methods is imperative for the diagnosis of these unusual pathogens.