Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 25, Num. 4, 2007, pp. 391-394

Indian Journal of Medical Microbiology, Vol. 25, No. 4, October-December, 2007, pp. 391-394

Brief Communication

Rapid detection of non-enterobacteriaceae directly from positive blood culture using fluorescent In situ hybridization

Wong EH, Subramaniam G, Navaratnam P, Sekaran SD

Department of Medical Microbiology, Faculty of Medicine, University Malaya, 50603, Kuala Lumpur

Correspondence Address: Department of Medical Microbiology (EHW, GS, SDS), Faculty of Medicine, University Malaya, 50603, Kuala Lumpur, Malaysia; and
School of Medicine and Health Sciences (PN), Monash University Malaysia, Jalan PJS 11/5, Bandar Sunway, 46150 Petaling Jaya, Malaysia. Email: shamalamy@yahoo.com.

Date of Submission: 18-May-2007
Date of Acceptance: 18-Jun-2007

Code Number: mb07106

Abstract

Keywords: Acinetobacter spp. , blood culture samples, fluorescently labeled probes

Nosocomial infections caused by gram negative bacilli have become an increasing problem worldwide. ^[1] This problem has escalated in tandem with the introduction and subsequent empirical administration of broad-spectrum antibiotics in hospitals. Multidrug resistant (MDR) A. baumannii and P. aeruginosa have been isolated as causative agents of a number of nosocomial infections including septicaemia, ventilator- and catheter-associated pneumonia, urinary tract and wound infections. ^[2] Among these, bloodstream infections are one of the main causes of death in hospitalized patients with mortality rates between 30 and 70%. ^[3] Thus, the rapid identification of the causative agent of septicemia is imperative in improving the overall prognosis of the patient. Routine laboratory diagnosis of positive blood cultures such as gram stain, biochemical tests and other standard bacteriological methods, all of which could take up to 48 h for an accurate identification of the pathogen to be made. In addition, culture and sensitivity assays to determine the antibiotic profile of the infecting agent could take up to an additional 24 h. Fluorescent in situ hybridization (FISH) has been used to detect various bacteria in clinical samples. ^{[4],[5],[6],[7],[8]} The general principle of this method is the use of a fluorescently labelled oligonucleotide probe that specifically hybridizes to the target sequence of 16S rRNA, thereby enabling visualization of the whole bacterium with a fluorescence microscope. The aim of this study was to evaluate the specificity and sensitivity of FISH for the identification of Acinetobacter spp. and Pseudomonas aeruginosa directly from blood cultures.

Materials and Methods

Blood cultures positive for gram negative bacilli were obtained from the Microbiology Diagnostic Laboratory, University of Malaya Medical Centre, Kuala Lumpur, Malaysia.

A direct gram stain was performed and identification of the organisms was done using standard laboratory methods and the API20NE System (bioMerieux SA, Marcy-l′Etoile, France). An aliquot of blood was simultaneously used in a modified FISH assay. ^[4] Briefly, 10-15 µL of aliquots from positive blood culture samples were applied onto glass slide, air-dried and fixed with 4% formaldehyde in 96% ethanol. The slides were dried and covered with 50 µL hybridization buffer [0.9 m NaCl, 20 mm Tris-HCl (pH 7.2), 0.1% SDS] containing labelled probes (10 ng/mL) prior to incubation at 50 °C for 40 min. The slides were then washed and dried prior to visualization using a fluorescence Carl-Zeiss microscope. Oligonucleotide probes, based on the 16S rRNA gene of the Acinetobacter spp. and Pseudomonas spp., were labelled with TAMRA and 6-FAM, respectively, at the 5´ end [Table - 1]. In addition, a universal eubacterial probe ^[9] was used as a positive control.

The probes were tested individually against various gram negative bacteria ( Vibrio cholera , Stenotrophomonas maltophilia , Bulkholderia cepacia , Chromobacterium violesceum , Escherichia coli , Klebsiella pneumoniae) to test their specificity. Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 15308 were included as positive controls. The sensitivity of Acinetobacter spp. and Pseudomonas spp. probes, respectively, were determined using a pure bacterial culture that was spiked into uninoculated blood culture bottles (Bactec Plus/Paeds Plus culture vial aerobic/anaerobic; Becton Dickinson, Heidelberg, Germany) in dilutions of between 10 ¹ and 10 ⁸ CFU/mL, which were then incubated 16-20 h at 37 °C. Viable bacterial counts in the blood culture media was determined before and after incubation.

Results

Both the probes were highly specific and hybridized to the respective target genus only and not to other bacterial genera [Figure - 1]. All the bacterial strains tested could be detected using the universal eubacterial probe. Microscopic sensitivity testing with serially diluted bacterial suspension revealed a limit of detection by FISH at 10 ³ CFU/mL. Thus, in our study, the sensitivity and specificity of FISH for the detection of Acinetobacter spp. and Pseudomonas spp. were 100%, respectively.

Discussion

Pseudomonas aeruginosa and Acinetobacter baumannii are the most frequently isolated non-fermentative gram negative species from critically ill and immunocompromised patients in intensive care units. Early detection of these microorganisms in clinical samples and blood can result in more definitive antimicrobial therapy. FISH is a suitable method for rapid and specific detection of pathogenic bacteria in clinical samples without time-consuming cultivation. The entire assay took less than 2 h compared to the conventional laboratory methods that require 1-3 days, resulting in a time gain of almost 70 h. Besides that, this technique delivers additional information concerning cell count and cell morphology and is an in situ means of differentiation of mixed infections. This could allow for an early detection of microorganisms and thus more definitive antimicrobial treatment of the infected patients could be adjusted 1 or 2 days earlier. This in turn could reduce the overall mortality among patients with gram negative bacteraemia as has been documented in previous studies. ^[10] FISH has proven to be a powerful molecular method for identification, visualization and quantification of organisms of interest in microbial communities. ^[11] Several reports show that FISH has already been successfully applied for the detection of E. coli , H. pylori, Staphylococcus aureus and Brucella spp . ^{[5],[6],[7],[8]} However, this is the first report on detection of acinetobacters and Pseudomonas aeruginosa using FISH in Malaysia.

Several studies have documented the value of molecular techniques, including PCR for amplification and detection of microbial DNA or RNA in order to identify bacteria in clinical specimens. ^[12],[13] Although PCR is a highly sensitive technique that can be used in direct identification of bacteria in blood, ^[12],[13] it may not be appropriate for daily routine work as it is time-consuming, expensive and expertise demanding compared to FISH, which is rapid and inexpensive. Furthermore, the added advantage of FISH over PCR is that extraction of DNA from bacteria is not required in the former.

In conclusion, the ability of rapid and simultaneous detection of non- Enterobactericeae in clinical samples within 2 h without time-consuming cultivation and identification by standard bacterial techniques may suggest FISH as an alternative method in routine diagnostic laboratory.

Acknowledgement

This study was supported by IRPA grant 06-02-03-0109 PR 0047/19-06 and VOTE-F F0163/2005A from the Government of Malaysia and University of Malaya.

References

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