Septicemia is a life-threatening event which requires rapid appropriate therapy. As the outcome for patients with septicemia depends on factors including the septicemia-causing pathogen, rapid microbiological laboratory diagnosis is desirable (13
In fact, it is well established that a severe infection may proceed to a systemic inflammatory response syndrome that may culminate in septic shock (17
). While in the late phase of these events, immunomodulatory therapy, including anti-inflammatory cytokines or cytokine antagonists as well as coagulation inhibitors and antioxidants, is essential for therapy, appropriate antimicrobial therapy is decisive in the early phase (32
). Clearly, rapid identification of the sepsis-causing pathogen is a prerequisite for early appropriate antimicrobial treatment.
As conventional laboratory methods require 1 to 3 days before microorganisms grown in blood cultures can be identified, we wanted to evaluate the practicability, sensitivity, and specificity of FISH for identification of microorganisms from blood cultures. For this purpose, a 16S, 18S, and 23S rRNA-based approach was developed, as 16S, 18S, and 23S rRNA have been extensively used to elucidate the phylogenic relationships of bacteria on the inter- and intragenic levels (2
). Moreover, 16S rRNA targets have been used successfully for diagnostic PCR and FISH assays (2
; Trebesius et al., submitted).
Testing of the specificities of the various oligonucleotide probes revealed that the probes were highly specific; they hybridized to the desired target strain only and not to related microorganisms. By using a set of oligonucleotides that would theoretically allow identification of ca. 95% of the microorganisms most frequently recovered from blood cultures, we were in fact able to identify 111 of 115 microorganisms grown in blood cultures from septicemic patients on the genus and/or species level within ca. 2.5 h after a blood culture was flagged positive by an automated continuous-reading blood culture system. Thus, depending on the group of microorganisms investigated (bacteria, yeasts, etc.), a time saving of 26 to 46 h was achieved by FISH compared with conventional laboratory methods used for identification. Thus, antimicrobial treatment of these patients could be adjusted 1 or 2 days earlier.
This was particularly important when Gram stain examination revealed gram-positive cocci. In that case, we were able to segregate S. aureus
from coagulase-negative staphylococci by FISH. Although coagulase-negative staphylococci are the most prevalent bacteria (ca. 35%) in blood cultures, it is well established that in more than 90% of these cases the bacteria are contaminants from the normal skin flora (25
). Therefore, if coagulase-negative staphylococci can be identified immediately, unnecessary or inappropriate antimicrobial therapy can be avoided. On the other hand, the S. aureus
-specific probe also hybridized to methicillin-resistant S. aureus
(two specimens), which was particularly helpful in the screening of patients with known methicillin-resistant S. aureus
In the case of streptococci, the probes included in this study allowed differentiation between, e.g., S. pneumoniae
and enterococci. This allowed treatment with penicillin G or ampcillin plus gentamicin, respectively, to be selected earlier. However, a probe specific for E. faecium
, which is often resistant to conventional antibiotics, does not exist. Unfortunately, a probe that would hybridize to all viridans group streptococci could not be designed, as this group is phylogenetically heterogeneous. Therefore, using the set of oligonucleotides described herein, it is not possible to segregate viridans group streptococci from other pathogens with similar morphologies, such as Pediococcus
, although these species occur only rarely in blood cultures (Table ) (30
If gram-negative rods were found by Gram stain examination, we were able to distinguish among P. aeruginosa
, S. maltophilia
, and Enterobacteriaceae
. This is also important, as infections caused by the three types of bacteria should be treated with different antimicrobial agents. E. coli
is the most frequent gram-negative bacterium recovered from blood cultures (Table ) (30
). However, there is no probe available that is completely specific for E. coli
. The most E. coli
-specific probe that we have tested was still reactive with Shigella
spp. Although Shigella
spp. do not play a significant role, if any at all, in patients with septicemia, we have not included this probe in the present study.
Infections with yeasts, e.g., C. albicans
, C. glabrata
, or C. krusei
, could be recognized by the probes selected. This fact might be important for a differential treatment of fungemia. Thus, it is known that, e.g., C. krusei
shows intrinsic resistance against fluconazole (20
The microorganisms grown in four blood culture specimens containing Propionibacterium
spp., M. osloensis
, Lactococcus lactis
, and Bacteroides
spp., respectively, could not be identified on the genus level with the set of oligonucleotide probes used in this study. On the other hand, these bacterial species are rarely recovered from blood culture and do not play important roles in septicemia. Nevertheless, it is possible to include oligonucleotide probes specific for, e.g., Bacteroides
spp. for FISH of blood cultures (2
Recently, the direct identification of intestinal bacteria in blood by means of PCR and Southern hybridization has been demonstrated (11
). Although this approach was highly sensitive (detection of 10 to 100 microorganisms per 0.3 ml of blood), it may be associated with several problems, including specificity. Thus, if DNA is detected, it is unclear whether it actually represents live invading microorganisms or simply dead presorbed microorganisms, or microorganisms engulfed in and killed by polymorphonuclear leukocytes. Moreover, the method is time-consuming, expensive, and not appropriate for daily routine work (23
). FISH, in contrast, is a rapid, cheap, and reliable method.
Similar to our study, direct identification of bacterial isolates in blood cultures by chemiluminescent DNA probes that detect the rRNAs of certain target organisms has been reported previously (5
). However, the method used in the present study is easier and more rapid. Similar to the study referenced above, we feel that the costs of FISH may be justified by what might be saved in unnecessary antimicrobial therapy and possibly a shortened hospital stay. Moreover, reduction of unnecessary antimicrobial treatment not only results in a reduction of expenditures for antimicrobials but also in increased antimicrobial susceptibility of the microorganisms accounting for nosocomial infections (33
In summary, we have demonstrated that FISH is a rapid and reliable method for direct identification and differentiation of bacteria grown in blood cultures. FISH is rapid, cheap (about $20 per positive blood culture specimen), valid, and appropriate for daily routine work. In our institution, one technician is sufficient for performing all necessary examinations. Because of the simple technical protocol, there are no special equipment or facilities required for performing FISH. Nevertheless, ongoing studies in our institution need to elucidate whether an earlier pathogen identification by FISH actually results in earlier appropriate antimicrobial therapy and in a better clinical outcome for the patients.