|Year : 2012 | Volume
| Issue : 4 | Page : 490-495
|Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory tract specimens by real time PCR
Arash Ghodousi1, Bizhan Nomanpour1, Setareh Davoudi2, Parviz Maleknejad1, Maryam Omrani1, Nasim Kashef3, Taghi Zahraei Salehi4, Mohammad Mehdi Feizabadi1
1 Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Infectious Disease, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Microbiology, Faculty of Biological Sciences, Tehran, Iran
4 Department of Microbiology, Faculty of Veterinary Medicine, Tehran, Iran
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|Date of Web Publication||4-Mar-2013|
| Abstract|| |
Staphylococcus aureus is a significant cause of hospital-acquired pneumonia (HAP), particularly in mechanically ventilated patients. We used the fibronectin-binding protein A gene (fnbA) for the species-specific and quantitative detection of S. aureus directly from lower respiratory tract (LRT) specimens by a Taq Man real time PCR. For this reason, a total of 269 lower respiratory tract (LRT) specimens collected from patients with hospital-acquired pneumonia were assayed. Ampliﬁcation of fnbA in serial dilutions ranged from 10 9 CFU/ ml to 10 2 CFU/ml. Standard curve of triplicate every dilution had slope 3.34 ± 0.1 and R 2 > 0.99 with SD 0.1. Based on these data, the sensitivity and specificity of the newly developed real time PCR targeting the fnbA gene were both 100%. The Cohen's Kappa test showed the Kappa value of 1.0. The fnbA gene is a potential marker for the species-specific detection of S. aureus and can be used to detect this bacterium in any clinical specimens by real time PCR. Moreover, this method reduces the time needed for quantitative detection of Staphylococcus aureus from LRT specimens to nearly 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.
Keywords: fnbA Gene, real time PCR, respiratory infection, Staphylococcus. aureus
|How to cite this article:|
Ghodousi A, Nomanpour B, Davoudi S, Maleknejad P, Omrani M, Kashef N, Salehi TZ, Feizabadi MM. Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory tract specimens by real time PCR. Indian J Pathol Microbiol 2012;55:490-5
|How to cite this URL:|
Ghodousi A, Nomanpour B, Davoudi S, Maleknejad P, Omrani M, Kashef N, Salehi TZ, Feizabadi MM. Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory tract specimens by real time PCR. Indian J Pathol Microbiol [serial online] 2012 [cited 2020 Sep 19];55:490-5. Available from: http://www.ijpmonline.org/text.asp?2012/55/4/490/107787
| Introduction|| |
Staphylococcus aureus is a major human pathogen involved in nosocomial respiratory tract infections in health care settings, particularly in critical care units. , This bacterium is one of the first pathogens to colonize the respiratory tract of hospitalized patients. It plays a limited role in community-acquired pneumonia (one exception being post-influenza infection), whilst it assumes paramount importance in hospital-acquired pneumonia (HAP), particularly in mechanically ventilated patients. , Pathogenesis of S. aureus is contributed to the expression of cell surface protein receptors designated microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that interact specifically with proteins present in the host plasma and extracellular matrix. , Fibronectin-binding proteins (FnBPs) A and B are two of the multifunctional MSCRAMMs of S. aureus, which recognize fibronectin, fibrinogen, and elastin. Surface expression of FnBPs by S. aureus is a virulence determinant that permits this pathogen to colonize host tissues and alter the infective process.
S. aureus is responsible for 20-30% of HAP episodes, but the recent emergence of methicillin-resistant as an increasingly common pathogen in all forms of pneumonia (HAP, VAP, community- and health care-associated pneumonia) is very threatening and needs an accurate method for rapid diagnosis. ,
Bacteriological techniques such as culture on various media and biochemical tests are routine procedures for isolation of S. aureus from respiratory specimens such as broncho-alveolar lavage (BAL). However, these methods are time-consuming and can threaten the life of patients with HAP due to the long time required for identification and enumeration of causative agents. Moreover, culture-based techniques have lower sensitivity, specificity, and rapidity than molecular techniques. 
In accordance to guidelines for management of HAP issued by American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA), either semi-quantitative or quantitative culture data can be used for the management of patients with HAP. It was also later added that, ''Quantitative cultures increase specificity of the diagnosis of HAP without deleterious consequences." , However, the culture-based enumeration method is commonly semi-quantitative and requires at least 3 days yielding results.  Moreover, the results obtained by this method are not always consistent and reproducible.  False negative cultures are another concern with the bacteriologic approach, mainly due to recent prescription or change in antibiotic therapy, especially in the preceding 24 h, but up to 72 h can lead to progress of disease and its complication.  This is not possible in all situations. On the other hand, molecular methods represent alternatives combining high sensitivity, specificity, speed, and reliability for identification. Several nucleic acid amplification-based assays have been reported for S. aureus that are based on specific gene targets as well as universal sequences, offering different advantages and limitations. ,,, Thus, additional reliable tools for detection of S. aureus are of major interest.
Real time PCR seems to be a sensitive and quick method for the detection and enumeration of infectious agents in clinical specimens including those collected from patients with HAP. , In such conditions, this procedure takes less than 2 hours and produces results with high specificity and sensitivity.
The main aim of this study was to develop a highly sensitive, specific and also rapid method for direct detection and quantification of S. aureus from patients with HAP.
| Materials and Methods|| |
Clinical Specimens and Isolates
A total of 269 LTR specimens including broncho-alveolar lavage (BAL), mini-BAL, and endotracheal aspirates were collected from 3 main teaching hospitals in Tehran, Iran. These specimens were collected sequentially from patients at ICU during December 2009-December 2010.
From every LRT specimen, 2 ml was kept at -20° C for real time PCR reaction, and the remaining was used for bacterial culture and identification using standard methods. In brief, a calibrated loop (0.5 ml) was used to streak the specimens on sheep blood agar, and the colonies were counted after overnight incubation at 37° C.  Identification of isolates as S. aureus was based on Gram staining, colonial morphology, and production of catalase. The suspected colonies were further tested for biochemical reactions including, manitol fermentation, and coagulase production.
The reporting points for culture and counting the CFUs for the physicians were 1000-10,000-50,000 and 100,000. ,
A multiple alignment in NCBI was done to find oligonucleotide sequences specific for S. aureus. Conserved sites were identified, and a primer pair and probe were designed using primer3 plus ( http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi ). The specificity of the candidate primers and probe for all bacterial sequences in the database were verified by FASTA analysis ( http://www.ncbi.nlm.nih.gov/GenBank ).
For amplification of the S. aureus fibronectin-binding protein A (FnbA) gene,forward primer: 5-AGTGAGCGACCATACAACAG-3 (20 bp), reverse primer: 5-CATAATTCCCGTGACCATTT-3(20 bp), and probe: 5-FAM-AAGCACAAGGACCAATCGAGG-BHQ-1- 3 (21 bp, T = 63.0) were designed.
Real Time PCR
DNA was extracted using enzyme digestion and the phenol chloroform method. In Brief, 200 uL of sample in standard counting tubes were suspended in 200 uL TEN buffer (100 mM EDTA, 0.15 M NaCl, 100 mM Tris PH 7.5). The solution was then centrifuged at 4,000 rpm for 5 min, and the sediment suspended in 250 uL of lysis buffer I (lysostaphin 1 mg/ml, lysozyme 25 mg/ml, and 12 mM Tris) and incubated in 37° C for 30 minutes. Then, the second lysis buffer (1% SDS and 20 mg/ml proteinase K in TEN buffer) was incubated for 10 min at 70° C. Subsequently, the lysate was treated with phenol/chloroform. DNA was precipitated with cold isopropanol. The DNA sediment was washed with 70% ethanol and re-suspended in 50 uL sterile TE buffer (5 Mm EDTA, 10 Mm Tris-HCl PH 7.6) after evaporation.  Five micro liter aliquots of extracted DNA solution were used as templates in real time PCR.
Standardization of Real-time PCR
S. aureus ATCC 29213 was used as positive control and bacterial DNA from Pseudomonas aeruginosa (ATCC 49189), Stenotrophomonas maltophilia (NTCC10257), Acinetobacter baumannii (NCTC 19606), Klebsiella pneumoniae (NTCC 5056), Escherichia More Details coli (NTCC 21157), Streptococcus pneumoniae (D39), Streptococcus oralis (NTCC 57342), Streptococcus mitis (NTCC 58274), Legionella pneumophila (NTCC 11192), Mycoplasma pneumoniae (NTCC 10119), and human DNA were used as negative controls in real time PCR. Beta actin gene (primer and probe) was used as an internal control with every sample to detect PCR inhibitor as recommended.  The human white blood cell DNA was used as negative control in this study since our primers and probe do not react with human genome.
To make standards need for real time PCR, DNA was extracted from the positive control strain of S. aureus ATCC 29213. The amount of extracted DNA was measured by a Nano drop (Analytik Jena-Germany). Conversion of the mass to molecules was done using this formula:  Mass (in grams) × Avogadro's number/Average mol. wt. of a base × template length = Molecules of DNA
For double-stranded templates, we used 660 gm/mole/base.  To generate a standard curve, we prepared eight 10-fold dilutions, starting with 1 × 10 9 template copies and ending with 100 copies. This range of dilution clinically is significant and reflects the linearity of standard curve due to limitation of FAM dye excitation at this range.
The specificity of the developed real time PCR was assessed using bacterial isolates and samples that contained various gram-negative bacilli and gram-positive cocci as well as yeasts isolated from different sources. To further check the sensitivity and specificity of our real time PCR assay, we tested 230 clinical isolates of staphylococci including S. aureus (n = 137), S. epidermidis (n = 21), S. saprophyticus (n = 16) and other CoNS (n = 56) and also 49 veterinary isolates of staphylococci including S. aureus (n = 28) and S. intermedius (n = 21) in the experiment.
For plotting standard curve, real time PCR assays were performed in triplicate.
Real Time PCR Reaction
Amplification and detection were performed in LinGene K Real Time PCR apparatus (Bioer, Hangzhou, P.R. China). The reactions consisted of 0.2 mM of each primer and probe, 0.2 mM dNTP, 5 mM MgCl 2 , 1 U of DNA polymerase (mi Taq) (Metabion, Martinsried, Germany), 5 ul of 10 mM PCR buffer, and 5 ul of the template DNA in total volume of 45 ul with double distilled water. The cycling program was adjusted for 10 min at 95° C and then 35 cycles of 20 s each at 95°C (denaturation) followed by 55 s at 59.4° C with fluorescent collection (annealing and extension). Every sample was run in triplicate, and the mean was reported.
Comparisons of culture and real time PCR was done by using Cohen΄s Kappa test. The SPSS 13.0 software program was used for Chi-square and confidence interval analysis of the data. We used the slope of the standard curve in the equation: E = 10 (- 1/slope] –1 for calculating efficacy of PCR (E).  The slope can be calculated using the formula: Y = slope X + b. In this formula, Y is cycle threshold, X is log 10 template amount, and b is Y-intercept. The Y-intercept is an indication of the sensitivity of the assay and how accurately the template has been quantified. 
| Results|| |
Culture and Characterization of Isolates
Of 269 LRT specimens, 51 yielded S. aureus in culture [Table 1]. Using culture, co-infections of S. aureus with other organisms were detected in 58% of specimens (n = 24) [Table 2]. Totally, polymicrobial growth was observed in 16.9% of all specimens (n = 34). Counting of S. aureus colonies demonstrated less than 10 3 to more than 10 5 CFU/ml in positive cultures [Table 2].
|Table 1: Results of qualitative culture from LRT specimens collected from patients with HAP|
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|Table 2: The number of CFUs for S. aureus detected by quantitative culture and real time PCR from LRT specimens and polymicrobial infection in patients with hospital-acquired pneumonia at 3 main teaching hospitals in Tehran|
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Real Time PCR
Real time PCR found 51 samples as positive for S. aureus. The same result was obtained from culture. Standard curve of triplicates had a slope of 3.34 ± 0.1 and R 2 > 0.99 with SD 0.1. Efficacy of the PCR test was 0.999 (E = 10 (-1/slope] - 1).  Results of real time PCR and quantification of every sample is shown in [Table 2].
Based on the obtained data, the specificity and sensitivity of the newly developed real time PCR targeting the fnbA gene for 269 LRT specimens was 100%. Amplification of target genes from S. aureus in serial dilutions ranged from 10 2 to 10 9 CFU/ml. The technique could detect every calculated tube count with SD <1%. The results were confirmed in triplicate.
Moreover, Cohen's Kappa test using SPSS (version 13.5) showed the result of culture and real time PCR were almost in perfect agreement (Kappa value 1.0, standard error 0.00, with P. Value of 0.001)
Prevalence of fnbA in Non-S. aureus Bacteria
To analyze the specificity of the newly designed FnbA-specific probe and primers, the prevalence of the fnbA gene among 93 non-S. aureus staphylococci isolated from patients and also 49 veterinary isolates of staphylococci was investigated at the DNA level. The real time PCR presented here showed 100% specificity; i.e., the fnbA was not detected in non-S. aureus staphylococci.
In addition, all other gram-negative bacilli and gram-positive cocci included in this study were negative in PCR targeting fnbA.
| Discussion|| |
Staphylococcus aureus is one of the most common nosocomial pathogens in health care settings, particularly in critical care units. From epidemiologic and pathogenic point of view, the clinical entity of pneumonia due to S. aureus has evolved over time.
The prosperity of S. aureus as a pathogen can, in part, be contributed to the expression of cell surface protein receptors designated microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that interact specifically with proteins present in the host plasma and extracellular matrix.  The actions of MSCRAMMs as virulence factors allow S. aureus to adhere to the host cells surface and to damage tissue and help it to avoid phagocytosis by neutrophils. ,, Two of the multifunctional MSCRAMMs of S. aureus are fibronectin-binding proteins (FnBPs) A and B, which recognize fibronectin, fibrinogen, and elastin. ,,,, In current study, the fnbA was studied as a diagnostic target for specific identification of S. aureus by real time PCR. Despite the homology of N-terminal region of fnbA with fnbB, a particular region of fnbA gene were used to design primers and a probe for species-specific detection of our target organism. The specificity of the primers were double-checked against a panel of S. aureus strains and other gram-positive cocci and gram-negative coccobacilli as well as yeast in the LRT specimens [Table 1]. The specificity was 100% as determined by blasting in ENTREZ database. Importantly, the newly developed real time PCR could detect genetically distinct strains as determined previously by PFGE. 
Identification of causative agents in disease states is simplified by the use of real-time PCR assays. These assays provide a fast and specific method to identify bacterial pathogens in sepsis and pneumonia, viral agents in hepatitis and respiratory diseases. ,,, In current study, fnbA as target for real time PCR detection of S. aureus showed high sensitivity (100%) and specificity (100%). It could detect and enumerate 100 bacteria directly from clinical specimens. We detected 100 bacteria in our standard specimen, and the range of detection directly from LRT specimens was between 275 CFU/ml in specimen number 32 to 6.24E + 09 CFU/ml in specimen number 31. Moreover, our real time PCR assay counted more CFU/ml than quantitative culture. The sensitivity of culture and CFU counting is less than real time PCR since the latter technique can detect uncultivable S. aureus, which normally cannot be detected due to antibiotic therapy pressure.
Many studies have targeted two or more genes including sa442 and nuc genes for detection of S. aureus in positive blood culture with high sensitivity (100%) and specificity (100%).  This is more expensive than our methods due to high cost of two sets of probe and primers. In another study, McDonald et al. used nuc and plv genes.  Using orfX gene as a target has given a high rate of false positive samples and decreased the specificity to 86%.  In another study, Wellinghausen et al. used nuc gene for rapid detection of S. aureus bacteremia by real-time PCR in whole blood samples that showed low sensitivity and specificity. 
Due to this fact that S. aureus strains negative for nuc, sa442 and pvl is reported and the presence of fnbA in all 58 sequenced S. aureus in NCBI database, this gene would be a potential marker for the species-specific detection of S. aureus and could be used to detect this bacterium in any clinical specimens by real time PCR and can result in substantial cost savings. Due to this fact that S. aureus is one of the leading cause of hospital-associated infections, if a diagnosis can be provided earlier and more reliably (higher sensitivity and specificity), patients who require anti-microbial therapy will receive it sooner. As well, less auxiliary testing should be required (e.g. additional infectious diseases tests such as cultures), and patients should have less morbidity and, therefore, fewer costs related to supportive therapy (e.g., intensive care related to a delay in diagnosis of sepsis). Providing a negative result sooner can have important implications for the over-prescription of antibiotics. For example, if a real-time PCR test result can more quickly rule out the pathogen compared with a culture-based method, then the clinician may be less inclined to use empirical antibiotics or if empirical antibiotics are used, the duration of treatment may be shortened, and this is the fact that inappropriate empiric antibiotic therapy in the intensive care units (ICUs) is a factor for promoting drug resistance. , The current approach would help in rapid and accurate diagnosis of S. aureus causing HAP and help to prescribe appropriate antibiotic. Knowing the etiologic agent of pneumonia as S. aureus is very critical in our hospitals since 71% isolates recovered from our patients with pneumonia are resistant to methicillin (unpublished data).
We recommend using real time PCR for patients with severe nosocomial pneumonia or other nosocomial infections in place of the time-consuming culture-based approaches.
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Mohammad Mehdi Feizabadi
Department of Microbiology, School of Medicine, Tehran University of Medical Science, Tehran
Source of Support: This work was supported by Tehran University of Medical Sciences, project No. 8740, Conflict of Interest: None
[Table 1], [Table 2]
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