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Infectious Diseases Unit
Laboratories of Molecular Epidemiology Microbiology
Department of Epidemiology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
ABSTRACT
Misidentification of coagulase-negative staphylococci (CoNS) may delay appropriate treatment. We investigated 20 clinical isolates identified as Kocuria spp. by the Vitek 2 system. All were identified as CoNS by 16S rRNA gene sequencing (18 Staphylococcus epidermidis, 1 Staphylococcus haemolyticus). Four Kocuria isolates were shown to be identical to CoNS from the same patient by pulsed-field gel electrophoresis. Isolates identified by Vitek 2 as Kocuria most likely represent misidentified CoNS, and if clinically indicated, should be investigated further by genomic methods.
TEXT
Coagulase-negative staphylococci (CoNS) are increasingly important human pathogens, often involved in infections related to implanted medical devices, such as intravenous catheters, prosthetic heart valves, and orthopedic implants (9). Correct identification of CoNS by microbiology laboratories facilitates early recognition and management of these infections, often necessitating removal of the infected device. The Vitek 2 system (Vitek Systems, bioMerieux, St. Louis, Mo.) is an automated bacterial identification and susceptibility testing system that uses fluorescence-based technology. This system was previously reported to correctly identify 90% of CoNS to the species level (8). With the introduction of the Vitek 2 system into our microbiology laboratory, we noticed a dramatic increase in the number of clinical isolates identified as Kocuria spp. Our investigation of one case of continuous bacteremia with Kocuria spp. revealed all isolates to be identical by pulsed-field gel electrophoresis (PFGE); isolates were subsequently identified as Staphylococcus epidermidis by 16S RNA gene sequencing (3). We hypothesized that phenotypic variation, a feature of pathogenic S. epidermidis (10), may cause misidentification by standard laboratory procedures. These findings prompted us to review the clinical and microbiological features of isolates identified as Kocuria spp. by our microbiology laboratory. In order to concentrate on clinically significant isolates, only isolates from blood or implanted medical devices were studied.
Sequential clinical isolates, identified as Kocuria spp. by Vitek 2 using the ID-GPC card were collected. Antimicrobial susceptibility profiles were determined by Vitek 2. An investigation was conducted in each case, with the aim of determining the clinical significance of the index isolate.
Growth on Congo red agar (CRA) has previously been noted to enable morphological characterization of phenotypically variable staphylococci (4, 5). Colonial morphology was studied on Trypticase soy agar plates containing 5% sheep blood (Hy-Labs, Rehovot, Israel) and on homemade CRA plates containing 0.01% Congo red (4, 5). Plates were incubated at 35°C with CO2 (5%) for 48 h and at room temperature for another 48 h.
PFGE analysis was performed on all available clinical isolates of Kocuria spp., and on any CoNS isolated from the same patient, to determine genetic relatedness. DNA preparation and cleavage were performed as described previously (2). Chromosomal restriction fragments were separated in a 1.0% SeaKem LE agarose gel (FMC BioProducts, Rockland, Maine) with the CHEF-DR III apparatus (Bio-Rad, Hercules, Calif.). Electrophoresis conditions were 6 V/cm for 22 h at 14°C, with pulse times ranging from 5 to 40 s. DNA was stained with ethidium bromide, photographed (Bio-Rad Gel Doc 2000), and analyzed using diversity-fingerprinting software (Bio-Rad).
For 16S rRNA gene amplification, DNA samples were prepared using a Wizard genomic DNA purification kit according to the manufacturer's instructions (Promega, Madison, Wis.). The nearly complete sequence of the 16S rRNA gene was amplified by PCR with the conserved primers 8F (5'-AGAGTTTGATYMTGGCTCAG-3') and 1942R (5'-ACCTTGTTACGACTT-3'). Amplification was carried out using Vent DNA polymerase (New England BioLabs) and genomic DNA template. PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 30 cycles of 60 s at 95, 56, and 72°C and a final elongation step of 10 min at 72°C. PCR products were purified using a Promega Wizard PCR Preps kit (Promega) and sequenced using an Applied Biosystems (Foster City, Calif.) model 373A sequencer. Sequences were analyzed and compared using BLASTN software (http://www.ncbi.nlm.nih.gov/BLAST/) (1).
Twenty clinical isolates from seven patients were identified as Kocuria spp. by Vitek 2 (19 K. varians and 1 K. rosea); of these, 19 were available for further study (Table 1). Sources of Kocuria isolates were as follows: blood culture (12 isolates), pacemaker electrode (3 isolates), pacemaker pocket (2 isolates), and one isolate each from a pleural empyema, a central venous catheter, and an intraosseous nail of an Ilizarov external fixator. Five of the seven patients were judged to have a clinically significant infection involving an implanted medical device.
Eleven of 19 Kocuria isolates (58%) exhibited variability in colony morphology on CRA (Table 1). In addition, three patients also had CoNS blood isolates exhibiting colonial variability. Colonial variability manifested as colonies of different sizes (typically pinpoint and large colonies), various elevations above the agar (dome-shaped and flat colonies), and variable uptake of Congo red resulting in dark red or light pink colonies.
For all four patients who had CoNS isolated from blood (patients 1 to 4), PFGE patterns for the Kocuria isolates were identical to at least one of the CoNS. Of 19 available clinical isolates identified as Kocuria by Vitek 2, 18 were identified as S. epidermidis by 16S rRNA gene sequencing, and one was identified as Staphylococcus haemolyticus. Isolates from different patients were shown to be unrelated by PFGE (Table 1; Fig. 1).
The Vitek 2 system is not optimized for reporting Kocuria spp. The biochemical reactions used in the ID-GPC card are either negative or variably positive with the quality control strain of K. rosea (ATCC 186); none of the reactions included in this protocol yields a positive result in 95% or more of K. rosea strains (Vitek 2 ID-GPC quality control table, bioMerieux, 1999). All of the clinical isolates identified by Vitek 2 as Kocuria that we examined were in fact CoNS, 58% were phenotypically variable and most represented clinically significant infections associated with implanted medical devices. Although these results are uncontrolled, they suggest that identification of Kocuria by Vitek 2 may be a marker for invasive infection caused by CoNS. Phenotypic variation, manifesting as variability in colony morphology, is characteristic of virulent strains of S. epidermidis (10). It is possible that phenotypic variation is also responsible for misidentification of CoNS by biochemical methods, leading to erroneous reporting of CoNS as Kocuria by Vitek 2.
Clinical isolates reported by Vitek 2 as Kocuria spp. are not rare. Our laboratory, which serves a 1,200-bed hospital and processes about 47,000 blood cultures a year, identified 117 clinical isolates as Kocuria spp. between 1 December 2002 and 1 December 2003, among them 23 blood isolates and 21 vascular catheter isolates. If, as our data suggests, most of these isolates are in fact misidentified CoNS, then the problem presented here may be of significant magnitude.
Incorrect identification of CoNS by the microbiology laboratory increases the likelihood that a significant infection will be erroneously ascribed to contamination (6, 7). This problem may be addressed in several ways. Clearly, CoNS are best identified by genomic methods, such as 16S rRNA gene sequencing, thus eliminating confusion arising from phenotypic variation. However, high cost and the intensive labor required limit the use of such methods in clinical practice. Laboratories using the Vitek 2 system may wish to investigate the significance of isolates identified as Kocura spp., as we have done. Recognizing that Kocuria isolates frequently represent misidentified CoNS, it is reasonable to suppress reports of Kocuria by the laboratory and exchange them with "unidentified gram-positive cocci." In any case, a clinical specimen growing Kocuria should raise suspicion of CoNS infection, especially if the patient has an indwelling medical device and additional cultures are positive for CoNS. In these cases PFGE may be useful in demonstrating the genetic identity of different isolates, which strongly suggests true infection rather than contamination (6, 7). Finally, since phenotypic variation is known to be affected by the culture medium and growth conditions (4), the performance of the Vitek 2 system may improve when CoNS are cultured in conditions that minimize phenotypic variation.
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