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Year : 2007  |  Volume : 25  |  Issue : 4  |  Page : 391-394
 

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


1 Department of Medical Microbiology, Faculty of Medicine, University Malaya, 50603, Kuala Lumpur, Malaysia
2 School of Medicine and Health Sciences, Monash University Malaysia, Jalan PJS 11/5, Bandar Sunway, 46150 Petaling Jaya, Malaysia

Date of Submission18-May-2007
Date of Acceptance18-Jun-2007

Correspondence Address:
S D Sekaran
Department of Medical Microbiology, Faculty of Medicine, University Malaya, 50603, Kuala Lumpur
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.37346

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 ~ Abstract 

Fluorescent in situ hybridization (FISH) was carried out using two different oligonucleotide probes specific for Pseudomonas spp. and Acinetobacter spp. These probes were tested against different organisms and were found to be highly specific. Sensitivity testing showed that the probes were able to detect as low as 10 3 CFU/mL. In addition, FISH was carried out directly on positive blood culture samples and the detection of microorganisms took less than 2 h. We believe that FISH is a rapid method that can be used as a routine laboratory diagnostic technique for the detection of Acinetobacter spp. and Pseudomonas spp. in clinical samples.


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


How to cite this article:
Wong E H, Subramaniam G, Navaratnam P, Sekaran S D. Rapid detection of non-enterobacteriaceae directly from positive blood culture using fluorescent In situ hybridization. Indian J Med Microbiol 2007;25:391-4

How to cite this URL:
Wong E H, Subramaniam G, Navaratnam P, Sekaran S D. Rapid detection of non-enterobacteriaceae directly from positive blood culture using fluorescent In situ hybridization. Indian J Med Microbiol [serial online] 2007 [cited 2019 Jun 27];25:391-4. Available from: http://www.ijmm.org/text.asp?2007/25/4/391/37346


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 Top


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 L 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 L 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 More Details 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 Top


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 Top


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 More Details 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 Top


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 Top

1.National Nosocomial Infections Surveillance System. National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004. Am J Infect Control 2004; 32 :470-85.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: Microbiological, clinical and epidemiological features. Clin Microbiol Rev 1996; 9 :148-65.  Back to cited text no. 2    
3.Vincent JL, Bihari DJ, Suter PM, Bruining HA, White J, Nicolas-Chanoin MH, et al. The prevalence of nosocomial infection in intensive care units in Europe: Results of the European Prevalence of Infection in Intensive Care (EPIC) Study. JAMA 1995; 274 :639-44.  Back to cited text no. 3  [PUBMED]  
4.Jansen GJ, Moolbroek M, Idema J, Harmsen HJ, Welling GW, Degener JE. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes. J Clin Microbiol 2000; 38 :814-7.  Back to cited text no. 4    
5.Regnault B, Martin-Delature S, Lejay-Collin M, Lefevre M, Grimont PA. Oligonucleotide probe for the visualization of Escherichia coli / Escherichia fergusonii cells by in situ hybridization: Specificity and potential applications. Res Microbiol 2000; 151 :521-33.  Back to cited text no. 5    
6.Hartmann H, Stender H, Schafer A, Autenrieth IB, Kempf VA. Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescent in situ hybridization using peptide nucleic acid probes and flow cytometry. J Clin Microbiol 2005; 43 :4855-7.  Back to cited text no. 6    
7.Samarbaf-Zadeh AR, Tajbakhsh S, Moosavian SM, Sadeghi-Zadeh M, Azmi M, Hashemi J, et al. Application of fluorescent in situ hybridization (FISH) for the detection of Helicobacter pylori . Med Sci Monit 2006; 12 :CR426-30.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Welinghausen N, Nockler K, Sigge A, Bartel M, Essig A, Poppert S. Rapid detection of Brucella spp. in blood cultures by fluorescent in situ hybridization. J Clin Microbiol 2006; 44 :1828-30.  Back to cited text no. 8    
9.Amann RI, Krumholz L, Stahl DA. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J Bacteriol 1990; 172 :762-70.  Back to cited text no. 9    
10.Leone M, Bourgoin A, Cambon S, Dubec M, Albanese J, Martin C. Empirical antimicrobial therapy of septic shock patients: Adequacy and impact on the outcome. Crit Care Med 2003; 31 :462-7.  Back to cited text no. 10    
11.Amann R, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 1995; 59 :143-69.  Back to cited text no. 11    
12.Sabet NS, Subramaniam G, Navaratnam P, Sekaran SD. Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay. J Microbiol Methods 2007; 68 :157-62.  Back to cited text no. 12    
13.Yamamoto Y. PCR in diagnosis of infection: Detection of bacteria in cerebrospinal fluids. Clin Diagn Lab Immunol 2002; 9 :508-14.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]


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