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A hybrid sequence-specific oligonucleotide-ELISA method for rapid detection of bacteremia.

INTRODUCTION

Rapid detection of blood stream infections (BSIs) and appropriate antimicrobial treatment may facilitate more efficient management of patients with bacteremia and/or sepsis. Mortality rates related to sepsis remain high in spite of advances in pharmacologic agents, improvement in infection control and clinical management of patients (1). Inadequate antimicrobial therapy during first 24 hours of patient's admission results in a significant decline in survival (2, 3). Standard diagnosis of bacteremia mainly relies on culture-based testing, which requires at least 48 to 72 hours for completion. Although molecular methods based on nucleic acid testing (NATs) demonstrated more rapid diagnoses and are highly sensitive, blood cultures are still considered the gold standard for diagnosis of bacteremia. However, the NATs are routinely used to identify many microbial pathogens, and have provided a significant technological advantage for rapid identification of various microorganisms (4-6). Nonetheless, technically, some are complex and can be cumbersome in a clinical laboratory setting. This study describes a simple procedure allowing rapid identification of species specific bacterium from a mixed amplified sample, using patient's blood. The procedure consists of a hybrid assay based on PCR and ELISA format. Currently, this technique allows detection and identification of selected gram-positive bacteria in less than 5-6 hours and may provide a rapid, sensitive and cost effective detection method for identification of microorganisms commonly cultured from patient's blood diagnosed with bacteremia or sepsis.

MATERIALS AND METHODS

Bacterial strains and DNA isolation

The bacterial strains used in this study were obtained from hospital clinical laboratory at the University of Mississippi Medical Center. Bacteria were obtained in the form of isolated cultures on agar plates. Prior to DNA extraction, each sample was streaked on blood agar and was examined by Gram staining to confirm purity and Gram reaction. Overnight cultures were washed with chilled STE (100mM NaCl, 10mM Tris, 1mM EDTA) and quantified by spectrophotometer. Then the DNA was extracted using the instruction provided by the manufacturer (DNeasy kit; Qiagen, Valencia, CA). The extraction of DNA from blood was performed using a modification of the Purescript Capture Column method (Gentra Systems, Minneapolis, MN). Briefly, 400 [micro]l aliquot of whole blood was subjected to red blood cell lysis-buffer. Nucleated cells were recovered by centrifugation for 3 minutes at 10,000 rpm. The cell pellet was resuspended in 200 pl of lysis buffer (TE containing 50-100 [micro]g/ml of lysostaphin), then was applied to a capture column. The DNA extraction was performed according to the manufacturer's instruction.

Estimation of sensitivity

The sensitivity of bacterial detection by PCR in the blood samples was initially tested with S. aureus. S. aureus was grown in T odd-Hewitt medium to approximately 2.8 x [10.sup.6] colony forming units (CFU)/ml. Tenfold serial dilutions of the bacterial culture was prepared, and 50 [micro]l of each dilution was inoculated into 400 []l of healthy donor blood. Then 50 [micro]l of each blood sample was plated on blood agar plates to estimate the bacterial CFU in the blood. The sensitivity of bacterial detection by PCR in the blood samples was initially tested with S. aureus.

Bacterial cell counts via PCR

DNA was extracted from blood samples as described above. Prior to PCR analysis, reagents (buffer, nucleotides and primers) were filtered through a Centricon-100 centrifugal filter device (Millipore, Millipore Corporation), to reduce the amount of endogenous contaminating DNA. For PCR analysis, two primers (DG74 and 143SA) corresponding to a region within the 16S ribosomal RNA gene were used to amplify gram positive bacterial DNA (7). The same PCR protocol was utilized for both bacterial DNA extracted from cultures and from blood samples.

Evaluation of probe specificity

Probe specificity was tested by sequence specific oligonucleotide (SSO) hybridization (8). For detection of selected gram-positive bacteria, the DNA was amplified with group specific primers (Table 1) and hybridized to the SSO probes. The hybridization blot was prepared using 2 [micro]l each of the amplified products, blotted onto Nytran membranes using a 96-well manifold (8). The blots were hybridized with [sup.32]/P-labeled SSO-probes (9). Six sequence-specific probes (listed in Table 1) were used for identification of S. aureus; S. capitis; S. epidermidis; S. gp G; S. agalactiae; S. pneumoniae; S. gp C and E. faecalis. In addition, a positive control (S. pneumoniae) and a negative control were subjected to the same SSO hybridization analysis.

SSO-ELISA Assay

Maxisorb 96-well flat bottom plates (Nunc, Nalge Nunc International Corporation, USA. www.nuncbrand.com) were used to develop this assay. The plates were coated with 100 pl of Streptavidin (5 [micro]g/ml) per plate. To optimize probe concentration as well as to determine the sensitivity of probe sequence, two-fold decreasing serial dilutions of each probe (500-1 pmol), was immobilized in a 96-well flat bottom plate, wells (110), and wells 11 -12 were blanks. The biotin incorporated PCR product of bacterial DNA (total 50 [micro]l) was tested in the plate according to the manufacturer instruction provided in the ELISA kit (Invitrogen-BRL-Gibco), as previously described (10). Positive signals were detected based on the absorbance at 405 nm interpreted by a programmed ELISA reader (Dynex Technologies Inc. Chantilly, VA).

[FIGURE 1 OMITTED]

RESULTS

The sensitivity of detection of bacterial DNA in blood samples was examined using the generic amplification primers DG74 and 143SA. The amplification pattern of the S. aureus DNA extracted from blood of is shown in Figure 1. At 45 cycles, we observed amplification bands with decreasing density (samples 1-4), in ethidium bromide-stained agarose gel, with maximum detection at a dilution (1.38 x [10.sup.2]) corresponding to 12 CFU/[micro]l. Incorporating the dilution factors, it was evident that the 1 pl of DNA sample that was utilized for tubes 5-9 in PCR assay contained no bacterial DNA (Figure 1). The SSO hybridization pattern is shown in Figure 2.

[FIGURE 2 OMITTED]

Although probes such as S. epidermidis, S. aureus, and S. pneumoniae were very specific, S. capitis, S. gpG, S. gpC and S. agalactiae were slightly cross-reactive within the species specificity. The S. gp C was as equally as S. pneumoniae reactive with S. pneumoniae probe, whereas, S. pneumoniae was not reactive with S. gp C probe. The probe sensitivity was evaluated within an OD reading range of 1.3-4.5, of which less than 1.3 was indicative of a weak signal and 4.5 was a strong signal as compared to reactivity of the negative control (Figure 3). S. epidermidis, S. pneumoniae and S. gp C were most sensitive and were detectable at minimum SSO-probe concentration of about 15.63 pmol (well #6, OD of 1.45 and 1.68 respectively). S. agalactiae, S. gpG and E. faecalis (well #4, OD of 1.35 each) and S. aureus (well #3, OD of 1.9) were detectable at a level of 31.25 pmol and 125 pmol respectively. A summary of test result and orientation of the SSO-probes in the plate is given in Table 2. There was cross-reactivity between S. gp G, E. faecalis and S. agalactiae, however, intensity of color development was less within their respective detection threshold. S. gp C was crossreactive with S. pneumoniae but S. pneumoniae was very specific

[GRAPHIC OMITTED]

DISCUSSION

A hybrid SSO-ELISA technique described here was designed to detect and identify species specific gram-positive bacteria. This is a simple technique with the potential of detecting bacteria in clinical specimens including blood and body fluids and is highly suitable in the clinical laboratory setting. Current techniques for detection of bacteria in clinical specimens, although highly sensitive and rapid, require careful planning of amplification procedures as well as sophisticated instrumentation (6, 11). The limitation of this technique is the shelf life of the Streptavidin coated SSO probes, which is estimated to be about 3 months, as well as sensitivity of the SSO-probes that specifically captures virtually all strains of different species. Several laboratories reported the use of PCR-ELISA-based techniques for diagnosis of microorganisms, however, these techniques were developed for the identification of a single microorganism (12, 13). Our technique was adapted, based on the principles, introduced by NUNC, Nalge International, 2000. However, it has been utilized in the field of molecular biology and immunology. In fact, the Gen Trak Inc. adapted such a technique for identification of the HLA class II (DR and DQ) genotypes.

CONCLUSIONS

We have described a rapid, relatively, easy and highly sensitive method for detection and identification of clinically relevant gram-positive bacteria. This method allows species specific identification and with farther development of probes, it could allow unlimited numbers of organisms to be identified by this technique. In addition, the technique is simple and suitable in a clinical laboratory setting with the potential for automation, and integration into the laboratory electronic data-information system.

ACKNOWLEDGMENTS

This work was supported in part by a fund from the Department of Surgery at the University of Mississippi Medical Center. Additional support was also provided by the National Institutes of Health grant #AI4365.

REFERENCES

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(3.) Kumar A, Ellis P, Arabi Y, et al: Cooperative antimicrobial therapy of septic shock database research group: Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. Chest 2009; 136:1237-1248.

(4.) Reno, W.L., McDaniel, D.O., Turner, W.W., Williams, M.D. Polymerase chain reaction for the detection of bacteremia. The American Surgeon 2001: 67:1-5.

(5.) Barghouthi SA. A universal method for the identification of bacteria based on general PCR primers. Indian J Microbiol 2011; 51:430-444.

(6.) Afshari A, Schrenzel J, Leven M, Harbarth S. Benchto-bedside review: Rapid molecular diagnostics for bloodstream infection -a new frontier? Critical Care 2012; 16:222-233.

(7.) Greisen, K., Loeffelholz, M., Purohit, A., Leong, D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 1994; 32:335-351.

(8.) Rajalingam R, Mehra NK, Jain RC, Myneedu VP. Polymerase chain reaction-based sequence-specific oligonucleotide hybridization analysis of HLA Class II antigens in pulmonary tuberculosis: Relevance to chemotherapy and disease severity. J Infectious Dis 1996; 173:669-676.

(9.) McDaniel DO, Naftilan J, Barber WH. Limiting detection of an amplification signal for HLA-D region and VNTR genes by 32P-PCR. Biotechniques. 1993; 15:140-145.

(10.) Dave S, Carmicle S, Hammerschmidt S, Pangburn MK, McDaniel LS. Dual Roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and Factor H. J immunol 2004; 173-471-477.

(11.) Liesenfeld O, Lehman L, Hunfeld K-P, Kost G. Molecular diagnosis of sepsis: New aspects and recent developments European J Microbiol Immunol 2014; 4: 1-25. DOI:10.1556/EuJMI.4.2014.1.1

(12.) Morata, P., Queipo-Ortufil, M., Reguera, J., Garcia-Ordonez, M., Cardenas, A., Colmenero, J. Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis. J Clin Microbiol 2003; 144-148

(13.) Gillespie, BE, MatheGw, AG, Draughon, FA, Jayarao, BM, Oliveri, SP. Detection of Salmonella enterica somatic groups C1 and E1 by PCR-enzyme-linked immunosorbent assay. J Food Prot 2003; 66(12): 2367-2370

D. Olga McDaniel [1,2], Jason Guillot [2], Larry S. McDaniel [2,3], William W. Turner [1,2], Ross Fremin [1], Gita Subramony [1], Mark Williams [1,2]

[1] Department of Surgery, [2] School of Medicine, department of Microbiology University of Mississippi Medical Center, Jackson, Mississippi
Table 1. Amplification Primers and SSO-probes

ID       Specificity       Seauences

DG74     Universal         5'-agg-agg-tga-tcc-aac-cgc-a-3'
143SA    Universal         5'-gan-gac-gtc-aar-tcn-tca-tgc-3'
P1       S. epidermidis    5'-ccg-gtg-gag-taa-cca-ttt-gga-gct-3'
P2       S. aureus         5'-ccg-gtg-gag-taa-cct-ttt-agg-agc-3'
P3       S. gp G           5'-cgg-tga-ggt-aac-cta-tta-gga-gcc-3'
P4       S. agalactiae     5'-cgg-tga-ggt-aac-ctt-tta-gga-gcc-3'
P5       S. gp C           5'-cgg-tga-ggt-aac-cgt-aag-gag-cc-3'
P6       S. pneumoniae     5'-aac-tga-gac-tgg-ctt-taa-gag-att-a-'
P7       E. faecalis       5'-cgg-tga-ggt-aac-cct-ttt-gga-gcc-3'

Table 2. Microtiter plate orientation and sample identification

Probes            S. epi  S. aur  S. gp G   S. agal   s. gp C  S. pneum

Control             +       +        +         +         +        +
S. epidermitidis    +       -        -         -         -        -
S. aureus           -       +        -         -         -        -
S. agalactiae       -       -        -         +         -        -
S. gp G             -       -        +      [+ or -]     -        -
E. faecalis         -       -     [+ or -]  [+ or -]     -        -
S. pneumoniae       -       -        -         -         +        +
S. gp C             -       -        -         -         +        -
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Author:McDaniel, D. Olga; Guillot, Jason; McDaniel, Larry S.; Turner, William W.; Fremin, Ross; Subramony,
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Date:Apr 1, 2015
Words:2016
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