Patent Application
20060275795
This invention relates to the fields of Microbiology and Moleculary biology. Particularly, the present invention relates to methods of detecting bacterial pathogenicity and species identity based on genes that encode transcriptional regulators.
There is a need for the detection of bacteria pathogens that can cause diseases in mammals and plants. However, traditional methods to detect the presence of bacterial pathogens require an extended period of time for growing these bacteria from a background of competing microorganisms and an increase in bacterial cell numbers to more readily aid in identification.
For example, the standard FDA procedure for detection of Listeria in food products takes 4 days and the identification of Listeria colonies is done by eye (Bacteriological Analytical Manual, 7th Ed., 1992; Chapter 10). Other bacteria, such as Leptospires, take eight weeks or longer to grow in laboratory and have stringent nutritional requirements.
An alternative detection method is to use immunology-based assays, such as ELISA. The immunology-based procedures reduce or eliminate the requirement of a growth period, but are not very efficient in detecting low numbers of bacteria. Moreover, ELISA often relies on the use of bacteria of various serogroups as antigens, and therefore any serogroups that are not included in the antigen preparations may give false negative results.
Accordingly, there is an urgency to develop and apply screening tests with enhanced sensitivity and specificity for pathogenic bacteria in an effort to promptly contain and eliminate bacteria infections.
The development and application of molecular methods involving nucleic acid amplification (such as PCR) have enabled vast improvement in the laboratory detection and identification of bacteria. The deciphering of complete genomes of various bacteria pathogens also opens new avenues for improving the detection and potential control of these pathogenic bacteria.
The foregoing needs are met to a great extent by methods for detecting a pathogenic species of bacteria or pathogenic strains of a species of bacteria by polymerase chain reaction (PCR) using primers specific for the DNA sequence of genes that encode putative transcriptional regulators.
In one embodiment, a method for detecting the presence of Listeria monocytogenes in a sample comprises the steps of subjecting the sample to PCR amplification using primers designed to target a putative transcriptional regulator gene Imo0733; and detecting the presence of an amplification product of the Imo0733 gene as an indication of the presence of Listeria monocytogenes.
In other embodiments, methods for detecting the presence of Pasteurella multocida, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, or pathogenic Leptospira strains in a sample by subjecting the sample comprise the steps of PCR amplification using primers designed to target a putative transcriptional regulator gene and detecting the presence of an amplification product of the putative transcriptional regulator gene as an indication of the presence of Pasteurella multocida, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, or pathogenic Leptospira strains.
The practice of the embodiments described in further detail below will employ, unless other wise indicated, conventional methods of microbiology, molecular biology, and immunology within the skill of the art. Such techniques are explained fully in the literature. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
One aspect relates to the use of transcriptional regulator genes for specific detection and potential control of pathogenic bacteria including Listeria monocytogenes, Pasteurella multocida, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, and Leptospira.
Listeria monocytogenes is an opportunistic bacterial pathogen responsible for causing a significant proportion of human foodborne diseases worldwide. Pregnant women, neonates, immuno-suppressed individuals and the elderly are particularly prone to L. monocytogenes infections. The fact that it is found in a variety of food sources, such as vegetables, milk, cheeses, fish, meat and poultry products; and are can tolerate high concentrations of salt, extreme pH and temperature; are of particular concern to the food industry and public health regulatory agencies (Doyle, et al., J. Food Prot., 64:410-429 (2001)). Recent outbreaks of listeriosis due to contaminated foods have highlighted the importance of continuing surveillance of opportunistic pathogens such as L. monocytogenes in ready-to-eat food products (Robinson, et al., Encyclopedia of Food Microbiology (2000); Vasquez-Boland, et al., Clin. Microbiol. Rev., 14:584-640 (2001)).
Pasteurella multocida is a Gram-negative, nonmotile, facultatively anaerobic coccobacillus that forms part of the commensal flora in the oral cavity, upper respiratory and gastrointestinal tract of domesticated and wild animals. It is capable of producing septicemic or respiratory diseases in animals by infecting organs and tissues that have been previously weakened by stress, injuries or other microbial infections (Rimler, et al., Pasteurella and Pasteurellosis, (1989); Bisgaard, Zentbl. Bakteriol., 279 (1993)). This species is also an opportunistic pathogen to humans; bite and scratch wounds from pet animals such as cats and dogs can result in severe localized infections (Rimler, et al., Pasteurella and Pasteurellosis (1989); Frederiksen, Zentbl. Bakteriol., (1993)).
Staphylococcus aureus is a common, Gram-positive species that is pathogenic to both man and animals. Through generation of enterotoxins and superantigens, it can provoke severe immune responses in the host, resulting in some unique and occasionally fatal disease entities such as toxic-shock syndrome and staphylococcal scarlet fever (Robinson, et al, Encyclopedia of Food Microbiology, (2000)). Apart from being a leading source of gastroenteritis via contaminated foods (Le Loir, et al., Genet. Mol. Res., 2:63-76, (2003)), S. aureus has also been responsible for an increasing number of hospital-acquired infections due to its ability to acquire and develop resistance to antibiotics (Robinson, et al, Encyclopedia of Food Microbiology, (2000); Montesinos, et al., Infect. Control Hosp. Epidemiol., (2003); Strommenger, et al, J. Clin. Microbiol., 41:4089-4094 (2003)). In particular, the emergence of methicillin-resistant S. aureus (MRSA), first noted over two decades ago, has made it one of the most important human bacterial pathogens of modern times.
Streptococcus pyogenes (Group A streptococcus or GAS) is a Gram-positive, opportunistic bacterial pathogen that is transmitted via direct contact or respiratory droplets. Although it may exist in the respiratory tracts and skin of humans without causing obvious illness, this bacterium will rapidly multiply and spread in the host tissues in cases where host defenses become weak or defective, producing a variety of clinical diseases (Cunningham, Clin. Microbiol. Rev., 13:470-511 (2000); Schwartz, et al., Lancet, 336:1167-1171 (1990); Musser, et al., Emerging infections, pp. 185-218 (1998); Bemer, et al., Eur. J. Pediatr., 158:527-529 (2000)). In its acute form, S. pyogenes infection often appears as pharyngitis, scarlet fever, impetigo or cellulitis. Any delay in treating the acute S. pyogenes infection may result in a more systematic, invasive, toxigenic form of disease, with manifestations ranging from bacteremia to streptococcal toxic shock syndrome. In a rare, worst case of scenario, acute rheumatic fever or acute giomerulonephritis may develop as immune-mediated post-streptococcal sequelae (Cunningham, Clin. Microbiol. Rev., 13:470-511 (2000)). As invasive GAS infections have emerged as an increasingly important health concern worldwide, it is vital that improved diagnostic techniques are available for early diagnosis and prompt treatment of infections due to S. pyogenes.
The genus Enterococcus comprises a large number of Gram-positive bacterial species that are present in the gastrointestinal tract in humans and animals as normal flora, and also form an important part of the lactic acid bacteria in foods (Murray, Clin. Microbiol. Rev., 3:46-65 (1990); Jett, et al., Clin. Microbiol. Rev., 7:462-478 (1994); Franz, et al., Int. J. Food Microbiol., 88:105-122 (2003)). With their extraordinary ability to obtain genetic elements encoding virulence traits or antibiotic resistance from each other and also from other bacteria, enterococci, especially E. faecalis and E. faecium, have emerged as significant human pathogens in many parts of the world, causing bacteremia, endocarditis and other nosocomical infections. Of particular notice, E. faecalis alone accounts for 80-90% and E. faecium for 10- 15% of human enterococcal infections, with E. gallinarum, and E. casseliflavus being other clinically relevant enterococcal species (Murray, Clin. Microbiol. Rev., 3:46-65 (1990)).
The genus Leptospira represents a diverse group of spirochete bacteria with varying pathogenic potential. Being ubiquitous in the environment, Leptospira is found in a wide range of feral and domestic animals, which act as reservoirs for this zoonotic pathogen. Leptospiral infection in humans invariably results from direct or indirect contact with the urine of infected animals. Although human leptospirosis often presents as flu-like episodes with sudden onset of fever, headache and chills, failure to promptly undertake antibiotic treatment for the infection may lead to severe, sometimes deadly, renal, hepatic and pulmonary damage in patients (Levett, Clin. Microbiol. Rev., 14:296-326 (2001)). With its non-specific symptoms, leptospirosis has been largely unrecognized and neglected for a considerable length of time. However, following development of improved detection methodologies in recent decades, the presence of leptospirosis in man and animals is better documented. In fact, leptospirosis is now considered as an emerging infectious disease worldwide.
A method for detecting pathogenic bacteria such as with PCR amplification uses primers designed to target transcription regulators of the pathogenic bacteria. Transcriptional regulators are specialized DNA binding proteins that play an essential role in directing gene expression within bacteria for their adaptation and survival in different environmental conditions. Because different bacterial species and subspecies are able to adapt to different and sometimes highly specialized environmental niches, unique transcriptional regulators would be required for each group of bacteria. Therefore, it is likely that transcriptional regulators may be genus-, species-, or subspecies- specific, with potential for diagnostic applications.
In an embodiment, the method comprises the steps of identifying a transcriptional regulator or a putative transcription regulator gene in a bacteria strain of interest, subjecting a sample to PCR amplification using primers designed to target the transcriptional regulator gene, and detecting the presence of an amplification product of the transcriptional regulator gene as an indication of the presence of the bacteria strain of interest.
The transcription regulator or a putative transcription regulator can be identified by conducting a Blast search on the genomic sequence of the bacteria strain of the interest and selecting those transcriptional regulator genes that display no homology with other DNA sequences at GenBank. Oligonucleotide primers can be designed from the selected genes using commercially available software, such as the Primer3 software (Whitehead Institute for Medical Research, Cambridge, Mass.). The PCR amplification conditions can be optimized based on the specific primer sequences.
As is well-known to one skilled in the art, the sample can be a bacterial culture sample, a tissue sample, a body fluid sample, a food sample, or a field sample. In one embodiment, DNA is extracted from the sample and is then subjected to PCR amplification. In another embodiment, the sample is subjected to direct PCR amplification.
Since transcriptional regulators and other regulatory proteins are essential components in the regulation of RNA synthesis and gene expression within bacteria, they may be potentially useful targets for treatment and control purposes. Therefore, it is also within the scope of this invention to use virulence-specific Leptospira genes or their derivatives in the inhibition of growth, reduction of pathogenicity, treatment, and prevention of leptospirosis caused by pathogenic Leptospira species.
For example, one possible treatment strategy would involve using pharmaceutically active agent(s) that would inactivate or alter the function of one or more of the proteins encoded by the above listed genes, which would either kill the pathogenic Leptospira or render it susceptible to the host immune system. One possible vaccine strategy would involve altering one or more of the above listed genes or promoter(s) for one or more of the above listed genes such that expression of the encoded protein(s) would be completely disrupted or altered. The alteration or disruption of expression would render pathogenic Leptospira avirulent and effective as a live attenuated vaccine.
These strategies may be suitable for the control of any bacterial pathogen that has identifiable genes encoding transcriptional regulators which are specific to said bacterial pathogen or pathogenic strains of a bacterial species. Examples of such bacteria include, but are not limited to Listeria monocytogenes, Pasteurella multocida, Staphylococcus aureus, Streptococcus pyogenes, and Enterococcus faecalis.
The commercial value of these methods lies in their applicability for rapid and specific laboratory detection and diagnosis of a broad spectrum of pathogenic bacteria. This overcomes the deficiency in the prior art by broad specificity for all instead of a limited few pathogenic species.
The following publications are incorporated herein by reference:
Liu, D., et al., Journal of Medical Microbiology 52:1065-1070 (2003); Liu, D., et al., International Journal of Food Microbiology 9: 297-304 (2004); Liu, D., et al., Journal of Microbiological Methods 58, 263-267 (2004); Liu, D., Lawrence, M. L., et al., Letters in Applied Microbiology 40: 69-73 (2005); Liu, D., et al., Research in Microbiology 156:564-567(2005); Liu, D., et al., 156: 944-948 (2005) and Liu, D., et al., Canadian Journal of Microbiology 54, in press (2006).
The present invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and Tables are incorporated herein by reference.
Bacteria.
Listeria and other bacterial reference strains were obtained from the American Type Culture Collection (ATCC) or the National Collection of Type Culture (NCTC). Some environmental isolates were obtained by us (Erdenlig, et al., Appl. Environ. Microbiol., 65:2827-2832 (1999); Erdenlig, et al., J. Food Prot., 63:613-619 (2000)), and other food and clinical isolates were provided by Dr. Catherine Donnelly (Department of Nutrition and Food Sciences, University of Connecticut) and Dr. Robert Mandrell (United States Department of Agriculture-Agricultural Research Service, Albany, Calif.). A total of 52 Listeria strains were examined (Table 1). These included 30 L. monocytogenes, ten L. innocua, three L. grayi, two L. ivanovii, three L. seeligeri, and five L. welshimeri strains. Moreover, 15 other common gram-positive and -negative bacterial species were analyzed to verify the specificity of the PCR primers developed.
Extraction of Genomic DNA.
Genomic DNA was isolated from stationary phase cultures by phenol/chloroform extraction and isopropanol precipitation. L. monocytogenes or other bacterial species were grown on 5% sheep blood agar plates (TSA II, Becton Dickinson Microbiology Systems, Cockeysville, Md.), and several colonies were used to inoculate 25 ml of brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.). Cultures were incubated at 37° C. overnight with shaking, and bacteria were pelleted from the entire 25 ml and resuspended in 2.5 ml of 1×TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0) containing 2 mg/ml lysozyme (Sigma, St Louis, Mo.). Each tube was incubated at 37° C. for 30 min, and 250 μl of 10% SDS and 25 μl of 10 mg/ml proteinase K (Sigma) were added. After incubation at 56° C. for 2 hours, one volume of phenol/chloroform/isoamyl alcohol (25:24: 1) (Sigma) was added to each tube. Following centrifugation, the supernatant was transferred to a fresh tube, and one volume of isopropyl alcohol and 150 μl of 5 M NaCl were added. DNA was pelleted by centrifugation and washed with 3 ml of 70% ethanol. The purified DNA was resuspended in 1×TE, and DNA concentrations were determined spectrophotometrically at absorbances of 260 and 280 nm in a GeneSpec I (Hitachi Genetic Systems, Japan). Two micrograms of purified DNA from each bacterial strain was diluted in distilled water to 10 ng/μl for PCR analysis.
Identification of L. monocytogenes Specific Genes.
With the complete genomes of L. innocua strain CLIP (serovar 6a) and L. inonocytogenes EGD-e (serovar 1/2a) being available at GenBank (GenBank/EMBL accession numbers AL592022 and AL591824) (Glaser, et al. Science, 249:849-852 (2001)), a comparative genomic analysis was conducted using annotation data and BLAST searches to select gene(s) that would be unique to L. monocytogenes. Specific primers from the selected genes were designed with Primer3 software (Whitehead Institute for Medical Research, Cambridge, Mass.), and synthesized by Sigma Genosys (The Woodlands, Tex.).
PCR Amplification.
PCR amplification was performed in a 25 μl volume using a GeneAmp PCR System 2400 (Perkin Elmers, Foster City, Calif.). Each reaction mixture consisted of 0.5 U Taq DNA polymerase (Fisher Scientific, Houston, Tex.), 1× PCR buffer (containing 10 mM Tris-HCl pH 9.0, 50 mM KCl and 1.5 mM MgCl2), 50 μM dNTPs, 25 pmol each primer, and 15 ng (1.5 μl) of template DNA. Reaction mixture with no template DNA was included as a negative control. The cycling program consisted of 1×94° C. for 2 min.; 25×94° C. for 20 sec., 50° C. for 20 sec. and 72° C. for 45 sec.; and 1×72° C. for 2 min. After the completion of all cycles, 3 μl of 10× DNA loading buffer was added to each tube, and the amplified products were examined by 1.0% agarose gel electrophoresis. At least two replications were run on all reactions to ensure consistent results.
To confirm the amplified products from the L. monocytogenes strains were actually from the Imo0733 gene, the amplicons from nine of the strains (ATCC 19111, ATCC 19112, ATCC 19113, ATCC 19114, ATCC 19115, ATCC 19116, ATCC 19118, EGD, and ATCC 15313) were digested with EcoNI and BpmI. The digested PCR products were observed by agarose gel electrophoresis to determine if the digests yielded bands consistent with the predicted sizes based on the EGD genome sequence.
The specificity of the PCR assay was further assessed by Southern blot hybridization. Briefly, PCR products from three L. monocytogenes strains and seventeen other species were separated by agarose gel electrophoresis and transferred to a nylon membrane (Hybond N+, Amersham Pharmacia, Piscataway, N.J.) using a standard protocol (Ausubel, et al., Current Protocols in Molecular Biology, (1994)). The 453 bp Imo0733 amplicon from EGD was labelled using the ECL direct nucleic acid labelling and detection system (Amersham Pharmacia Biotech) and used to probe the membrane. Prehybridization, hybridization, and stringency washes were all performed in tubes at 42° C. according to the manufacturer's protocol.
The detection limit of the PCR assay was determined using the conditions described above with serial dilutions of the 10 ng/μl genomic DNA stock from L. monocytogenes EGD (NCTC 7973) as the template. DNA quantities tested consisted of 10 ng, 1 ng, 0.1 ng (100 pg), 0.01 ng (10 pg), 0.001 ng (1 pg) and 0.0001 ng (0.1 pg) per reaction. An equal volume (1.5 μl) of template DNA was added to each reaction.
Results.
After comparison of the genomes of L. innocua strain CLIP (serovar 6a) and L. monocytogenes EGD-e (serovar 1/2a), one L. monocytogenes specific gene (Imo0733) was selected. This gene is located between nucleotide sequences 123783-124307, and encodes a 169 amino acid protein similar to a transcriptional regulator (Glaser et al., Science, 294:849-852 (2001)). Two L. monocytogenes specific oligonucleotide primers (Imo0733F: 5′CGCAAGAAGAAATTGCCATC-3′ (SEQ ID NO:1) and Imo0733R: 5′-TCCGCGTTAGAAAAATTCCA-3′ (SEQ ID NO:2)) were designed from the coding sequence of this gene. These primers correspond to the /mo0733 gene sequences at nucleotide positions 123844-123863 and 124277-124296, respectively, and allow amplification of a 453 bp DNA fragment by PCR.
Using the L. monocytogenes specific primers (Imo0733F and Imo0733R), the predicted 453 bp fragment was amplified from genomic DNA from all 30 L. monocytogenes strains regardless of serotypes or origins (Table 1 and
The detection threshold of these PCR primers under the described conditions using genomic DNA of L. monocytogenes grown in BHI broth was approximately 10 pg of DNA (
The Imo0733 amplicons from nine of the L. monocytogenes strains were confirmed by digestion with EcoNI and BpmI. According to the L. monocytogenes EGD genome sequence, digestion with EcoNI should yield 322 bp and 131 bp fragments, and digestion with BpmI should yield 213 bp and 240 bp fragments. All of the amplicons from the nine strains yielded bands at the predicted sizes from both enzymes (data not shown).
Southern hybridization using the Imo0733 amplicon from strain EGD as a probe demonstrated that the PCR products amplified from ATCC 19114 and ATCC 15313 were from the same gene, and it also demonstrated that the Imo0733 PCR product was not detectable from other species, including other Listeria species (data not shown).
Taken together, these results show that application of PCR primers (Imo0733F and Imo0733R) derived from Imo0733 gene resulted in the amplification of a specific 453 bp fragment from L. monocytogenes DNA only, which suggests that this gene is not present in the other four Listeria species as well. This assay provides an alternative means of rapidly and precisely diagnosing listeriosis due to L. monocytogenes because it is based on a novel transcriptional regulator gene unique to L. monocytogenes instead of relying on rRNA genes, iap, or the virulence gene cluster.
In addition, these results suggest that Imo0733 may be an important virulence gene regulator that modulates expression of genes that allow L. monocytogenes to adapt to a human host.
Bacteria.
A collection of 45 bacterial strains/isolates, including 10 Pasteurella multocida, 5 Mannheimia haemolytica, and 30 other bacterial species, were analysed (Table 2). These bacteria were either acquired from the American Type Culture Collection (ATCC) or the National Collection of Type Culture (NCTC), or isolated from clinical samples in our laboratory. Bacterial strains were initially grown on 5% sheep blood agar plates (TSA II, Becton Dickinson Microbiology Systems, Cockeysville, Md.) and transferred to flasks containing 25 ml of brain heart infusion (BHI) broth for batch cultures (Difco Laboratories, Detroit, Mich.). Bacterial cultures were incubated at 37° C. overnight with rotary aeration.
Preparation of Bacterial DNA.
Bacterial DNA was prepared using the method described in Example 1.
Identification of P. multocida Specific Transcriptional Regulator Genes.
The nucleotide sequences of P. multocida genes encoding transcriptional regulators were retrieved from the published P. multocida genome sequence (May et al, 2001), and BLAST searches were conducted to select those transcriptional regulator genes that display no homology with other DNA sequences at GenBank. Oligonucleotide primers were designed from these genes with Primer3 software (Whitehead Institute for Medical Research, Cambridge, Mass.) and synthesized by Sigma Genosys (The Woodlands, Tex.).
PCR Amplification.
PCR amplification was conducted in a volume of 25 μl using a GeneAmp PCR System 2400 (Perkin Elmer, Foster City, Calif.). The reaction mixture consisted of 0.5 U Taq DNA polymerase (Fisher Scientific, Houston, Tex.), 1× PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 1.5 mM MgCl2), 50 μM dNTPs, 25 pmol primers each and 15 ng DNA. Reaction mixture with no template DNA was used as a negative control. The cycling programs consisted of 1 cycle of 94° C. for 2 min.; 25 cycles of 94° C. for 20 sec., 60° C for 20 sec. and 72° C. for 45 sec.; and a final incubation at 72° C. for 2 min. After completion of all cycles, 3 μl of 10× DNA loading buffer was added to each tube, and the amplified products were examined by 1.0% agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg/ml). The stained gels were visualized under UV light, and results were recorded using a Chemilmager 5500 (BSI, Stafford, Tex.).
Results.
Upon comparison of P. multocida transcriptional regulator gene sequences (May et al, 2001) with other DNA sequences at GenBank via BLAST searches, two genes (Pm0762 and Pm1135) with no apparent homology to published DNA sequences were selected for further evaluation (Table 3). Oligonucleotide primers Pm0762F (SEQ ID NO:3) and Pmo0762R (SEQ ID NO:4) were designed to amplify a 567 bp DNA fragment by PCR, and primers Pm1135F (SEQ ID NO:5) and Pm1135R (SEQ ID NO:6) were designed to produce 489 bp product (Table 3).
The specificity of these primers was assessed with a collection of 45 bacterial strains/isolates, including 10 P. multocida, 5 M. haemolytica, and 30 other bacterial species (Table 2). As expected, both primer sets formed specific PCR products of appropriate size from genomic DNA of P. multocida only, but not from DNA of other bacterial species (Table 2 and
These results show that Pm0762 and Pm1135 are species-specific genes in P. multocida. The application of these primers (Pm0762F/R and Pm1135F/R) would therefore offer an additional means of rapidly and precisely identifying P. multocida.
Bacteria.
Bacterial reference strains were acquired from the American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC). Other strains were either isolated in this laboratory from clinical samples or obtained from external sources. A collection of 63 bacterial strains/isolates were analysed in this study. These included 14 S. aureus, 3 other Staphylococcus species, and 46 other bacterial species (Table 4). Bacterial strains were cultivated on 5% sheep blood agar plates (TSA II, Becton Dickinson Microbiology Systems, Cockeysville, Md.), and batch cultures were grown in of brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) at 37° C. with rotary aeration. Extraction of bacterial DNA.
Bacterial DNA was prepared using the method described in Example 1.
Identification of S. aureus Specific Transcriptional Regulator Genes
The nucleotide sequences of S. aureus genes encoding transcriptional regulators were retrieved from the published genome sequences of S. aureus (Kuroda et al, 2001), and BLAST searches were conducted to select transcriptional regulator genes demonstrating no homology with other gene sequences in GenBank. Oligonucleotide primers were designed from the transcriptional regulator genes unique to S. aureus with Primer3 software (Whitehead Institute for Medical Research, Cambridge, Mass.) and synthesized by Sigma Genosys (The Woodlands, Tex.).
PCR Amplification.
PCR amplification was performed in a volume of 25 μl using a GeneAmp PCR System 2400 (Perkin Elmer, Foster City, Calif.). The reaction mixture consisted of 0.5 U Taq DNA polymerase (Fisher Scientific, Houston, Tex.), 1× PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 1.5 mM MgCl2), 50 μM dNTPs, 25 pmol primers each and 15 ng DNA. Reaction mixture with no template DNA was used as a negative control. The cycling programs consisted of 1 cycle of 94° C. for 2 min.; 25 cycles of 94° C. for 20 sec., 60° C. for 20 sec. and 72° C. for 45 sec.; and a final incubation at 72° C. for 2 min. After completion of all cycles, 3 μl of 10× DNA loading buffer was added to each tube, and the amplified products were examined by 1.0% agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg/ml). The stained gels were visualized under UV light and results recorded using a Chemilmager 5500 (BSI, Stafford, Tex.).
Results.
After comparison of S. aureus transcriptional regulator genes (Kuroda et al, 2001) with other DNA sequences at GenBank via BLAST searches, two genes (i.e., Sa0836 and Sa0856) that encode putative transcriptional regulators were selected for further evaluation (Table 5). These genes appeared to be unique because they displayed no apparent homology with previously published DNA sequences. Oligonucleotide primers (Sa0836F (SEQ ID NO:7) and Sa0836R (SEQ ID NO:8)) were derived from Sa0836 that facilitated amplification of a 573 bp amplicon, and primers derived from Sa0856 (Sa0856F (SEQ ID NO:9) and Sa0856R (SEQ ID NO:10)) yielded a band of 599 bp by PCR (Table 5).
The specificity of these primers was evaluated with a collection of 63 bacterial strains/isolates containing 14 S. aureus, 3 other Staphylococcus and 46 other species (Table 4). Primers derived from these two genes generated PCR products of expected sizes from genomic DNA of S. aureus only, and not from DNA of other bacterial species (
These results indicate that S. aureus genes (Sa0836 and Sa0856) encoding putative transcriptional regulators are species-specific.
Bacterial Strains.
A collection of 60 bacterial strains comprising 10 Streptococcus pyogenes, 16 non-pyogenes streptococci, and 34 other Gram-positive and -negative bacteria was examined in the study (Table 6). Of these, the reference strains were obtained from the American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC); Streptococcus pyogenes human isolates were originated from Department of Microbiology, University of Alabama at Birmingham, Ala.; Staphylococcus aureus human isolates from Veterans Affairs Medical Center, Jackson, Miss.; other clinical/food bacterial strains were mostly isolated at College of Veterinary Medicine, Mississippi State University, Mississippi State, Miss. except for Mannheimia haemolytica D139 that was provided by Dr. Robert Briggs of National Animal Disease Center, Ames, Iowa.
Genomic DNA.
Bacterial DNA was prepared using the method described in Example 1.
Identification of S. pyogenes Specific Gene(s).
S. pyogenes genes that encode transcriptional regulators were obtained from the genome sequence of a M1 GAS strain SF370 (Ferretti, et al., Proc. Natl. Acad. Sci. USA, 98:4658:4653 (2001)), and screened against other DNA sequences at GenBank by BLAST searches. Only gene(s) uniquely present in S. pyogenes were selected for further evaluation. Oligonucleotide primers were then designed from the gene(s) of interest with Primer3 software (Whitehead Institute for Medical Research, Cambridge, Mass.), and synthesized by Sigma Genosys (The Woodlands, Tex.).
PCR Amplification.
PCR was conducted in a 25 μl volume using a GeneAmp PCR System 9600 (Perkin Elmer). The reaction mixture (25 μl) comprised 0.5 U Taq DNA polymerase (Fisher Scientific, Houston, Tex.), 1× PCR buffer (containing 10 mM Tris-HCI pH 9.0, 50 mM KCl and 1.5 mM MgCl2), 50 μM dNTPs, 25 pmol primers each and 10 ng DNA. The reaction mixture with no template DNA was used as a negative control. The cycling programs consisted of 1×94° C. for 2 min., 30×94° C. for 20 sec., 55° C. for 20 sec. and 72° C. for 45 sec., and 1×72° C. for 2 min. fter completion of all cycles, 3 μl of 10× DNA loading buffer was added to each tube, and the amplified products were examined in 1.0% agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg/ml). The stained gels were visualized under UV light and photographed using a Chemilmager 5500 (BSI, Stafford, Tex.).
Based on the results form BLAST searches, a S. pyogenes specific gene (Spy1258) that encodes a putative transcriptional regulator was identified from the genome sequence of a M1 strain SF370 (GenBank accession No. AE006565) (Ferretti, et al., Proc. NatL Acad. Sci. USA, 98:4658-4663 (2001)). It was noted that a stretch of nucleotides identical to Spy1258 (nt. 6651-7193) was also found in the complete genomes of S. pyogenes M3 strains MGAS315 (GenBank accession No. AE014154) (Beres, et al., Proc. Natl. Acad. Sci. USA, 99:10078-10083 (2002) and SSI-1 (GenBank accession No. AP005144) (Nakagawa, et al. Genome Res., 13:1042-1055 (2003)) as well as M18 strain MGAS8232 (GenBank accession No. AE010045) Smoot, et al., Proc. Natl. Acad. Sci. USA, 99:4668-4673 (2002)). However, the Spy1258 gene sequence was clearly absent in other bacterial genomes that are available at GenBank.
Therefore, a pair of oligonucleotide primers was designed from the putative transcriptional regulator gene Spy1258 (i.e., spy1258F: 5′-AAAGACCGCCTTAACCACCT-3′ (SEQ ID NO:11) and spy1258R: 5′-TGGCAAGGTAAACTTCTAAAGCA-3′ (SEQ ID NO:12)). These primers correlate to the Spy1258 gene sequence at nt 6686-6705 and nt 7092-7070, respectively, which facilitate the amplification of a 407 bp DNA fragment from S. pyogenes. Using these primers (spy1258F and spy1258R) in PCR with a collection of 60 bacterial strains, it was observed that a specific DNA fragment of the expected size (407 bp) was generated from all ten S. pyogenes strains only, but not from 16 non-pyogenes Streptococci (representing 7 separate species) and 34 other bacteria (Table 6 and
These results show that the putative transcriptional regulator gene Spy1258is specific for S. pyogenes, and it can be used as a diagnostic marker for rapid confirmation of Group A streptococci.
Bacterial Strains.
A collection of 88 bacterial strains including 22 E. faecalis, 11 E. faecium and 55 other Gram-positive and -negative bacteria were examined in the study (Table 7). Of these, the reference strains were obtained from the American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC); E. faecalis and E. faecium were cultured from seafood; and other bacteria were isolated from clinical specimens of human and animal origins.
Genomic DNA.
Bacterial DNA was prepared using the method described in Example 1.
Identification of E. faecalis Specific Gene(s).
E. faecalis genes that encode transcriptional regulators were retrieved from the genome sequence of a vancomycin-resistant E. faecalis strain V583 (Paulsen, I. T., et al., 2003, Science 299, 2071-2074) and screened against other DNA sequences at GenBank by BLAST searches. Only E. faecalis genes showing no obvious homology with other DNA sequences were selected for further evaluation. Oligonucleotide primers were then designed from the gene(s) of interest with Primer3 software (Whitehead Institute for Medical Research, Cambridge, Mass.), and synthesized by Sigma Genosys (The Woodlands, Tex.).
PCR Amplification.
PCR was performed in a 25 μl volume using a GeneAmp PCR System 9600 (Perkin Elmer). The reaction mixture (25 μl) consisted of 0.5 U Taq DNA polymerase (Fisher Scientific, Houston, Tex.), 1× PCR buffer (containing 10 mM Tris-HCl pH 9.0, 50 mM KCl and 1.5 mM MgCl2), 50 μM dNTPs, 25 pmol each forward and reverse primers and 10 ng DNA. The reaction mixture with no template DNA was included as a negative control. The cycling programs consisted of 1×9° C. for 2 min., 30×94° C. for 20 sec., 60° C. for 20 sec. and 72° C. for 45 sec., and 1×72° C. for 2 min. After completion of all cycles, 3 μL of 10× DNA loading buffer was added to each tube, and the PCR products were examined in 1.0% agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg/ml). The stained gels were then visualized under UV light and photographed by using a Chemiumager 5500 (BSI, Stafford, Tex.).
Results.
After comparison of E. faecalis transcriptional regulator genes with other DNA sequences at GenBank via BLAST searches, an E. faecalis specific gene (Ef0027, nucleotides 27614-28384) that encodes a putative phosphosugar-binding transcriptional regulator was selected from the sequence data of a vancomycin-resistant E. faecalis strain V583 (GenBank accession No. AF454824) (Paulsen, et al., Science, 299:2071-2974 (2003)). This gene appeared to be uniquely present in E. faecalis as it showed no homology with other microbial genomes that are available at GenBank. Therefore, forward and reverse oligonucleotide primers were designed from this gene (i.e., Ef0027F: 5′-GCCACTATTTCTCGGACAGC-3′ (SEQ ID NO:13) and Ef0027R: 5′-GTCGTCCCTTTGGCAAATAA-3′ (SEQ ID NO:14)). These primers correspond to the Ef0027 gene sequence at nt 27777-27786; and nt 28284-28265, respectively, which enable the production of a 518 bp fragment from E. faecalis DNA in PCR.
The specificity of the E. faecalis specific primers from the putative transcriptional regulator gene Ef0027 (i.e., Ef0027F and Ef0027R) was evaluated in PCR with a collection of 88 bacterial strains, including 22 E. faecalis, 11 E. faecium and 55 other Gram-positive and -negative bacteria (Table 7). It appeared that a specific DNA fragment of the expected size (518 bp) was amplified from E. faecalis strains only, but not from E. faecium and other bacteria as well as no DNA template control (Table 7 and
Bacterial Strains.
Leptospira and other reference bacterial strains were acquired from the USDA National Veterinary Services Laboratory (NVSL) or the American Type Culture Collection (ATCC). Twenty eight Leptospira strains representing seven pathogenic (ie, L. interrogans, L. alexanderi, L. borgpetersenii, L. kirschneri, L. kirschneri, L. noguchii, L. santarosai and L. weilii) and four non-pathogenic (ie, L. biflexa, L. inadai, L. meyeri and L. wolbachii) species were examined (Table 8). In addition, 46 other bacterial species/strains were included for assessment of the specificity of Leptospira primers developed (Table 9). Leptospira strains were cultured in EMJH broth supplemented with 10% rabbit serum (Difco Laboratories, Detroit, Mich.) and maintained by weekly subculture into fresh medium. For DNA isoaltion, Leptospira cultures were cultivated for seven days at 30° C. to stationary phase to a density of approximately 2×108 cells per ml. Cells were harvested by centrifugation, resuspended in 0.15 M PBS pH 7.2 and stored at −20° C. prior to DNA extraction. Other bacteria were initially grown on 5% sheep blood agar plates (TSA II, Becton Dickinson Microbiology Systems, Cockeysville, Md.), and batch cultures in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) were maintained at 37° C. with rotary aeration. Genomic DNA.
Bacterial DNA was prepared using the method described in Example 1.
Identification of Leptospira Specific Gene(s).
The gene sequences of L. interrogans serovar icterohaemorrhagiae strain Lai that encode transcriptional regulators were retrieved from the published genome data (Ren, et al., Nature, 422:888-893 (2003)), and BLAST searches were conducted to identify transcriptional regulator genes that demonstrate no homology with other gene sequences at GenBank. As a result, eleven genes encoding putative transcriptional regulators or hypothetic proteins (ie, La0825, la0954, la1937, la2032, la2640, la2894, la3133, la3152, la3231, la3825 and la4130) were selected from the large circular chromosome (CI) of L. interrogans serovar Icterohaemorrhagiae strain Lai (Table 10). Interestingly, transcriptional regulator gene la1937 is essentially the same as la2032 and la3152 in L. interrogans serovar Icterohaemorrhagiae (Ren et al., Nature, 422:888-893, 2003)). Primers were designed from the nine genes by using Primer 3 software (Whitehead Institute for Medical Research, Cambridge, Mass.), and custom synthesized (Sigma Genosys, The Woodlands, Tex.) (Table 10).
PCR Amplification.
PCR was performed in a volume of 25 μl using a GeneAmp PCR System 9700 (Perkin Elmer, Foster City, Calif.). The reaction mixture was made up of 0.5 U Taq DNA polymerase (Fisher Scientific, Houston, Tex.), 1× PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 1.5 mM MgCl2), 50 μM dNTPs, 25 pmol primers each and 15 ng DNA. A reaction mixture with no template DNA was used as a negative control in each run. The cycling programs consisted of 1 cycle of 94° C. for 2 min.; 30 cycles of 94° C. for 20 sec., 55° C. for 20 sec. and 72° C. for 45 sec.; and a final incubation at 72° C. for 2 min. After completion of all cycles, 3 μl of 10× DNA loading buffer was added to each tube, and the amplified products were examined in 1.0% agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg/ml). The stained gels were visualized under UV light and results recorded using a Chemilmager 5500 (BSI, Stafford, Tex.).
Results.
Using PCR primers from the nine putative transcriptional regulator or hypothetic protein genes (ie, La0825, la0954, la1937, la2640, la2894, la3133, la3231, la3825 and la4130) resulted in the amplification of specific products from 19 of the 24 Leptospira pathogenic strains (with the exception of L. interrogans serovar Ballum S-102, L. interrogans serovar Mini Szwajizak, L. interrogans serovar Tarassovi Perepelicin, L. interrogans serovar Sejroe, and L. santarosai serovar Sherrnani) (Table 8; data not shown). None of the primers above reacted with Leptospira non-pathogenic strains (ie, L. biflexa, L. inadai, L. meyeri and L. wolbachii), the other 44 common bacterial species/strains, or the negative (no DNA template) control in PCR (Table 8; data not shown).
These results show that the nine putative transcriptional regulator and hypothetic protein genes (ie, La0825, la0954, la1937, la2640, la2894, la3133, la3231, la3825 and la4130) are only present in pathogenic Leptospira species/strains, and hence can be used as markers for the detection of these bacteria strains. From a previous study with foodborne pathogen L. monocytogenes, it was noted that transcriptional regulator genes have some important roles to play in listerial virulence, since L. monocytogenes avirulent strains possess fewer such genes that virulent strains (Liu et al., J. Med. Microbiol., 52:1065-1070 (2003)). Therefore, similar to nonpathogenic Leptospira species, the five Leptosipra strains (ie, L. interrogans serovar Ballum S-102, L. interrogans serovar Mini Szwajizak, L. interrogans serovar Tarassovi Perepelicin, L. interrogans serovar Sejroe, and L. santarosai serovar Shermani) without the nine transcriptional regulator and hypothetic protein genes (ie, La0825, la0954, la1937, la2640, la2894, la3133, la3231, la3825 and la4130) may be much less virulent than the others containing these genes.
Aeromonas hydrophila ATCC
Bordetella avium 2-1178
Bordetella bronchiseptica
Clostridium perfringens
Escherichia coli ATCC
Enterococcus faecalis ATCC
Enterococcus faecium ATCC
Enterococcus avium ATCC
Enterococcus casseliflavus
Enterococcus durans ATCC
Enterococcus gallinarum
Enterococcus hirae ATCC
Enterococcus mundtii ATCC
Enterococcus raffinosus
Flavobacterium indologenes
Klebsiella pneumoniae ATCC
Listeria grayi ATCC 19120
Listeria innocua ATCC
Listeria ivanovii ATCC
Listeria monocytogenes EGD
Listeria seeligeri ATCC
Listeria welshimeri ATCC
Mannheimia haemolytica
Micrococcus sedentarius 2134
Pasteurella multocida ATCC 11039
Proteus vulgaris ATCC 13315
Pseudomonas aeruginosa ATCC
Salmonella typhimurium ATCC
Serratia marcescens ATCC 8100
Staphylococcus epidermidis ATCC
Staphylococcus aureus ATCC 25923
Staphylococcus cohnii 4850
Staphylococcus haemolyticus 2168
Staphylococcus hominus 1961
Staphylococcus saccharolyticus 956
Staphylococcus intermedius 840
Staphylococcus siuri 4856
Streptococcus canis 1001-3
Streptococcus dysgalactiae 814
Streptococcus equinus
Streptococcus equisimilis 834-4
Streptococcus pneumoniae
Streptococcus pyogenes ATCC 19615
Streptococcus uberis 815
Streptococcus zooepidemicus 861-1
Yersinia pseudotuberculosis
The nucleotide sequence of putative transcriptional regulator gene la1937 is nearly identical to those of la2032 and la3152; and thus primers from la1937 will also recognize la2032 and la3152.
The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any squence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.
This application claims priority from U.S. Provisional Application Ser. No. 60/660,332 filed Mar. 11, 2005. The entirety of that provisional application is incorporated herein by reference.
This invention was made with Government support under 58-6202-5-083 awarded by the U.S. Department of Agriculture. The Government may have certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60660332 | Mar 2005 | US |
