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Journal of Clinical Microbiology, September 2008, p. 3097-3100, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.00910-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Use of Fluorescence In Situ Hybridization for Rapid Identification of Staphylococci in Blood Culture Samples Collected in a Portuguese Hospital {triangledown} ,{dagger}

Ana Tavares,1,2,{ddagger} João Inácio,2 José Melo-Cristino,3 and Isabel Couto1,2*

UEI Micobactérias, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Rua da Junqueira, nr. 96, 1349-008 Lisbon, Portugal,1 CREM, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal,2 Hospital de Santa Maria, Instituto de Microbiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Egas Moniz, 1649-028 Lisbon, Portugal3

Received 12 May 2008/ Returned for modification 28 May 2008/ Accepted 10 June 2008


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ABSTRACT
 
Fluorescence in situ hybridization was used for the direct identification of staphylococci in blood cultures collected at a Portuguese hospital where staphylococci account for up to 35% of clinically relevant blood cultures. The assay was able to detect the presence/absence of staphylococci and distinguish Staphylococcus aureus from coagulase-negative staphylococci in 4.5 h.


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TEXT
 
Staphylococci—in particular, coagulase-negative staphylococci (CoNS)—are the pathogens most frequently isolated in blood cultures (2, 17). However, many of these CoNS are contaminants from skin flora, and their isolation from blood cultures may not correspond to infection (8). Thus, the rapid and reliable identification of staphylococci and the differentiation between Staphylococcus aureus and CoNS are essential to optimize diagnostic and therapeutic decisions.

Analysis of blood samples usually involves culturing in automated systems, followed by Gram staining, subculturing, and biochemical testing. Altogether, this procedure takes 24 to 48 h to provide identification after microbial growth detection. Methods that allow the direct identification of bacteria in positive blood cultures, such as fluorescence in situ hybridization (FISH), which detects nucleic acids within intact cells, are thus essential to overcome the delaying step of subculture (4, 10, 13, 14).

The purpose of the present study was to develop and evaluate the performance of a FISH protocol for the direct detection of staphylococci in positive blood cultures from a large teaching hospital in Lisbon (Portugal), where these bacteria account for up to 35% of all clinically relevant positive blood cultures.

The following oligonucleotide probes labeled at the 5' end with fluorochrome Cy3 (Thermo Hybaid, Ulm, Germany) were used for the FISH assays: EUB338 (5'-GCTGCCTCCCGTAGGAGT), targeting eubacteria (1); EUK516 (5'-ACCAGACTTGCCCTCC), targeting eukaryotes (1); and three additional 16S rRNA-targeted probes designed in this work, STA2 (5'-CATATCTCTGCGCATTTC) and STA3 (5'-GCACATCAGCGTCAGT), targeting Staphylococcus spp., and Sau66 (5'-AAGCTTCTCGTCCGTTCG), specific for S. aureus (Fig. 1). Probe specificity was evaluated by testing these sequences against 16SrRNA gene sequences available in GenBank.


Figure 1
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  		FIG. 1. Alignments of STA2, STA3, and Sau66 probes with the 16S rRNA gene complementary target sequences of Staphylococcus spp., M. caseolyticus, and E. coli. These sequences correspond, respectively, to nucleotides 686 to 716, 742 to 770, and 59 to 93 of the E. coli 16S rRNA (3). Mismatches are indicated by the highlighted cells.

Although several authors have referred to the advantages of working with peptide nucleic acid probes, in our work we felt no need to use these probes, which are considerably more expensive than oligonucleotide probes.

The optimization of the FISH experimental procedure, particularly cell lysis and the evaluation of probe specificity, was carried out with a set of reference strains obtained from the Culture Collection of the Molecular Genetics Laboratory of Instituto de Tecnologia Química e Biológica (ITQB/UNL): Staphylococcus aureus NCTC8325, Staphylococcus epidermidis ATCC 14990T, Staphylococcus saprophyticus ATCC 15305T, Staphylococcus haemolyticus ATCC 29970T, Staphylococcus lugdunensis ATCC 43809T, Staphylococcus hominis ATCC 27844T, Staphylococcus simulans ATCC 27848T, Staphylococcus warneri ATCC 27836T, Staphylococcus schleiferi subsp. schleiferi ATCC 43808T, Macrococcus caseolyticus ATCC 13548T, and Escherichia coli ATCC 35218. The yeast Cryptococcus neoformans PYCC3957T, from the Portuguese Yeast Culture Collection (PYCC), was also included as a positive control for the EUK516 probe. An additional group of 39 staphylococcal clinical isolates previously identified by biochemical and molecular techniques, which included S. aureus (22 isolates), S. epidermidis and S. haemolyticus (7 isolates each), and S. hominis (3 isolates) (12, 15, 16), was also included for probe evaluation.

For the FISH assays with the reference strains, cultures were grown to exponential phase and fixed with 4% paraformaldehyde as previously described (1). One microliter of fixed cell suspension was deposited in individual wells of 12-well Teflon glass slides (Superior Marienfeld, Lauda-Koenigshofen, Germany) and dried at 37°C. Before hybridization, the staphylococcal cells were permeabilized by one of the following protocols: (i) treatment with an enzyme mix of 750 µg/ml lysostaphin (Sigma Chemical Company, St. Louis, MO) and 5 mg/ml lysozyme (Sigma) for 1 h at 37°C (11); (ii) incubation with 1 mg/ml lysozyme for 10 min at 30°C, followed by 1 mg/ml lysostaphin for 5 min at 30°C (10); or (iii) incubation with 1 mg/ml lysozyme for 15 min at 37°C, followed by incubation with 10 µg/ml lysostaphin for 5 min at the same temperature (adapted from reference 9).

For each hybridization reaction, 10 µl of hybridization solution (20% formamide, 0.9 M NaCl, 20 mM Tris-HCl [pH 8.0], 0.01% sodium dodecyl sulfate) containing 30 ng of each probe was added to each well and the slides were incubated for 3 h at 46°C. Both the permeabilization and hybridization procedures were performed in a saturated humid chamber. Thereafter, the slides were incubated in 50 ml of prewarmed wash solution (0.215 M NaCl, 20 mM Tris-HCl [pH 8.0], 5 mM EDTA, 0.01% sodium dodecyl sulfate) for 15 min at 46°C and air dried in the dark. The results were observed using an Olympus BX50 microscope (Olympus Optical Co., Hamburg, Germany) equipped with a filter for Cy3.

Of the three permeabilization protocols tested, the one described by Kempf and colleagues (10) provided the best compromise between cell integrity and hybridization signal intensity. Although STA2 would theoretically hybridize in a region with higher accessibility to the 16S rRNA molecule (6), FISH assays using STA2 and STA3 probes with the reference strains demonstrated that the two probes hybridized equally well, with STA3 showing higher specificity toward the M. caseolyticus negative control, as would be expected from the in silico analysis (Fig. 1). Therefore, STA3 was used in the remaining assays.

The Sau66 probe hybridized only with S. aureus strains, showing no unspecific hybridization with the other staphylococcal species tested, except for a weak hybridization with the S. schleiferi subsp. schleiferi reference strain, as expected by the in silico analysis (Fig. 1) and also reported with other S. aureus probes (7). Because of the very low incidence of S. schleiferi subsp. schleiferi as a causative agent of sepsis, this cross-reactivity was not considered diagnostically significant.

The specificity of the hybridization signals obtained with these probes was further evaluated by hybridizing the same reference strains with the 18S rRNA-targeted universal eukaryote probe EUK516. As expected, only the C. neoformans cells emitted hybridization signals with this probe, indicating that no unspecific binding occurs between the probes used and the staphylococcal cellular matrix. Further testing of probes STA3 and Sau66 with the 39 staphylococcal isolates of clinical origin confirmed the correct hybridization of these probes to their target species.

The optimized FISH protocol using probes STA3 and Sau66 was then applied to the direct identification of staphylococci in 92 blood cultures obtained from the bacteriology laboratory of a 1,300-bed teaching hospital (Lisbon, Portugal), with medical, surgical, pediatrics, intensive care, and emergency medicine units, during June to October 2005. These corresponded to blood cultures from patients with suspected general bloodstream infection, which were inoculated in aerobic (BacT/Alert FA, bioMérieux, Marcy l'Etoile, France), anaerobic (BacT/Alert FN), or pediatric (BacT/Alert PF) blood culture bottles. This collection included 77 blood cultures identified in the clinical laboratory as containing staphylococci, 14 cultures containing other bacteria, and 1 culture with the yeast Candida parapsilosis. A sterile blood culture was also tested to evaluate the interference of culture medium components on the hybridization results.

There was a need to adapt the previously optimized FISH protocol for the analysis of blood cultures. In particular, due to the high density of the culture medium, which complicated cell homogeneous treatment and proper fixation to the slide, the 12-well slides were replaced by normal microscope slides, into which 10 to 20 µl of blood culture was streaked and air dried. There was no need to use specific reagents such as poly-L-lysine to increase the adherence of specimens to the slides. The cells were fixed with 1:1 ethanol-phosphate-buffered saline because of the simplicity of this protocol and because long storage of the samples before their analysis was not needed. The remaining experimental protocol followed the procedures previously optimized.

In general, the staphylococcal cells showed good hybridization signals, which allowed their rapid detection in the blood cultures, even in the samples with low cell concentrations (data not shown). We did not observe interference of the charcoal with the probes or autofluorescence from the medium components, with a few exceptions, which did not influence the interpretation of the FISH signals.

The FISH results correlated well with the identifications obtained at the hospital laboratory using conventional techniques, and only two discrepancies were found (Table 1). The STA3 probe hybridized correctly with the 77 Staphylococcus species isolates tested and with only 1 of the 15 nonstaphylococcal samples tested. This culture, previously identified as Enterococcus faecalis, contained a mixture with CoNS cells that were readily identified by FISH. The 22 positive blood cultures containing S. aureus, which included both methicillin-susceptible and methicillin-resistant S. aureus isolates, hybridized correctly with the Sau66 probe, whereas the remaining 70 non-S. aureus blood cultures showed no hybridization with this probe, except for a culture previously identified as containing S. haemolyticus, which turned out to be a polymicrobial culture. Thus, the rapid detection of polymicrobial or mixed cultures is another advantage of FISH assays (7).


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  		TABLE 1. Results of the direct identification of Staphylococcus spp. by FISH in blood cultures previously identified by conventional laboratory techniques

The Sau66 probe revealed high levels of sensitivity (100%) and specificity (98.6%), with positive and negative predictive values of 95.6% and 100%, respectively, for the identification of S. aureus. These results are similar to those reported by others (4, 7, 10, 13, 14).

Other groups have recently reported positive outcomes for the introduction of FISH-based assays in the routines of their clinical laboratories (5, 14). The study presented here is a pioneer in Portugal, where for the first time FISH was applied to the analysis of blood cultures in parallel to the routine analysis. The FISH protocol optimized in this study provided results for the presence/absence of staphylococci and the differentiation between S. aureus and CoNS within 4.5 h, which constitutes a time savings of 24 to 48 h over the conventional laboratory identification.

Due to the critical role played by the laboratory in the management of bloodstream infections in large hospitals such as the one in this study, where S. aureus and CoNS accounted for 18.9% and 16.1%, respectively, of all bacteria isolated from blood cultures during the year of sample analysis, rapid access to this information is of utmost importance. This method may constitute a valuable tool complementary to conventional laboratory analysis by providing a same-day result that can be used to streamline antimicrobial therapy, thus preventing excessive vancomycin usage, improving patient prognoses, and reducing the length of hospitalization (5).

Our experience showed that FISH is a rapid, reliable, and appropriate method for the daily work routine. Except for the epifluorescent microscope, the protocol does not involve special equipment. Also, with the exception of labeled probes, which are used in very low concentrations, the method does not involve expensive reagents. Additionally, the methodology does not require technical expertise, as most of the procedures are routine in the workflow of the microbiologic clinical laboratory. The total hands-on time is short (less than 1 h), thus not involving the need of extra personnel.

Based on the promising results obtained, we believe that in the future, FISH will become a valuable diagnostic tool for the direct identification of microorganisms in blood cultures that is complementary to conventional laboratory techniques.


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ACKNOWLEDGMENTS
 
This study was supported by Project FCG-61056, from Fundação Calouste Gulbenkian, Lisbon, Portugal, awarded to I.C. J.I. was supported by grant SFRH/BPD/18453/2004 from Fundação para a Ciência e a Tecnologia (FCT, Portugal).

We gratefully acknowledge Hermínia de Lencastre (Laboratory of Molecular Genetics, ITQB, UNL) for providing all the staphylococcal reference strains and clinical isolates used in this study. We also acknowledge Miguel Viveiros (IHMT, UNL) for critical comments.


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FOOTNOTES
 
* Corresponding author. Mailing address: UEI Micobactérias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira, nr. 96, 1349-008 Lisbon, Portugal. Phone: 351-21-3652652. Fax: 351-21-3632105. E-mail: icouto{at}ihmt.unl.pt Back

{triangledown} Published ahead of print on 18 June 2008. Back

{dagger} We dedicate this article to the memory of Prof. Isabel Spencer-Martins (1951-2008). Back

{ddagger} Present address: Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica, ITQB, Universidade Nova de Lisboa, Oeiras, Portugal. Back


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Journal of Clinical Microbiology, September 2008, p. 3097-3100, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.00910-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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