Multiplex PCR

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Multiplex PCR for the detection of AmpC beta-lactamase genes Abstract Oliognucleotide primers are provided that are specific for nucleic acid characteristic of certain AmpC beta-lactamases. The primers can be employed in methods to detect the presence or absence of an AmpC beta-lactamase gene in samples, and to identify nucleic acid characteristic of AmpC beta-lactamase genes in samples, particularly, in clinical isolates of Gram-negative bacteria. Background A disturbing consequence of the use, and over-use, of beta-lactam antibiotics (e.g., penicillins and cephalosporins) has been the development and spread of beta-lactamases. Beta-lactamases are enzymes that open the beta-lactam ring of penicillins, cephalosporins, and related compounds, to inactivate the antibiotic. The production of beta-lactamases is an important mechanism of resistance to beta-lactam antibiotics among Gram-negative bacteria. Expanded-spectrum cephalosporins have been specifically designed to resist degradation by the older broad-spectrum beta-lactamases such as TEM-1, 2, and SHV-1. Microbial response to the expanded-spectrum cephalosporins has been the production of mutant forms of the older beta-lactamases called extended-spectrum beta-lactamases (ESBLs). Although ESBL-producing Enterobacteriaceae were first reported in Europe in 1983 and 1984, ESBLs have now been found in organisms of diverse genera recovered from patients in all continents except Antarctica. The occurrence of ESBL-producing organisms varies widely with some types more prevalent in Europe (TEM-3), others more prevalent in the United States (TEM-10, TEM-12 and TEM-26), while others appear worldwide (SHV-2 and SHV-5). These enzymes are capable of hydrolyzing the newer cephalosporins and aztreonam. Studies with biochemical and molecular techniques indicate that many ESBLs are derivatives of older TEM-1, TEM-2, or SHV-1 beta-lactamases, some differing from the parent enzyme by one to seven amino acid substitutions. In addition, resistance in Klebsiella pneumoniae and Escherichia coli to cephamycins and inhibitor compounds such as clavalante have also arisen via acquisition of plasmids containing the chromosomally derived AmpC beta-lactamase, most commonly originating from Enterobacter cloacae, Citrobacter freundii, Hafnia alvei, and Morganella morganii. It is of particular concern that genes encoding the beta-lactamases are often located on large plasmids that also contain genes for resistance to other antibiotic classes including aminoglycosides, tetracycline, sulfonamides, trimethoprim, and chloramphenicol. Furthermore there is an increasing tendency for bacteria to produce multiple beta-lactamases. These developments, which occur over a wide range of Gram-negative genera, represent a recent evolutionary development in which common Gram-negative bacteria are availing themselves of increasingly complex repertoires of antibiotic resistance mechanisms. Clinically, this increases the difficulty of identifying effective therapies for infected patients. Organisms overexpressing AmpC beta-lactamases are a major clinical concern because these organisms are usually resistant to all the beta-lactam drugs except the dipolar ionic methoxyiminocephalosporins such as cefepime and cefpirome and the carbapenems. However, recently an Enterobacter cloacae clinical isolate associated high-level resistance to cefepime and cefpirome with overexpression of and a deletion within the ampC structural gene was reported. Barnaud et al., FEMS Microbiology Letters, 195:185-190 (2001). Overexpression of AmpC beta-lactamases can occur in two ways, the deregulation of the chromosomal gene expressing the AmpC beta-lactamase or the acquisition by gram-negative organisms of a transferable ampC gene either on a plasmid or other transferable element. The latter have commonly been called plasmid-mediated AmpC beta-lactamases. The ability to identify the difference between constitutive overexpression of AmpC beta-lactamase from the chromosome or a plasmid is important for hospital epidemiology. Organisms with inducible chromosomal ampC beta-lactamase genes include E. cloacae, E. aerogenes, Citrobacterfreundii, Morganella morganii, Hafnia alvei, Serratia marcescens, and indole positive Proteus spp. Strains of these organisms that overexpress the chromosomal genes are collectively called derepressed mutants. Therefore, by identifying the organism the laboratory can identify the ability of that organism to overexpress the AmpC beta-lactamase. Escherichia coli strains can also overexpress their chromosomal ampC beta-lactamase gene and are termed hyperproducing E. coli. Plasmid-mediated ampC genes are derived from the chromosomal ampC gene of several members of the family Enterobacteriaceae, such as E. cloacae, C. freundii, and others. But not all members of the family Enterobacteriaceae encode a gene for AmpC beta-lactamases or are the origin of plasmid-mediated genes, such as K. pneumoniae or E. coli, respectively. Therefore, the distinction between a plasmid-mediated AmpC beta-lactamase and the endogenous enzyme is difficult to determine in both hyperproducing E. coli strains and organisms with inducible chromosomal AmpC enzymes. This distinction, however, is critical for hospital infection control. Plasmid-mediated genes whether they are extended-spectrum beta-lactamases (ESBLs) or AmpC enzymes can spread rapidly to members of the same species or organisms of different genera. In addition, multiple beta-lactamases within one organism, such as multiple ESBLs or a combination of ESBLs and AmpC enzymes can make phenotypic identification of the AmpC enzyme difficult. Unfortunately, for these reasons, the detection of AmpC, particularly plasmid-mediated AmpC, beta-lactamase resistance goes undetected in most clinical laboratories. The ability to distinguish between different types of ampC beta-lactamase nucleic acid in a clinical sample is useful for such epidemiological purposes as identifying how particular bacteria of interest have spread, thus aiding in infection control. It is also useful for identifying the proper antibiotic treatment for a patient. Thus, there is a need for techniques that can quickly and accurately identify the particular types of beta-lactamases that may be present in a clinical isolate or sample, for example. This could have significant implications in the choice of antibiotic necessary to treat a bacterial infection. Summary of the invention The present invention is directed to the use of oligonucleotide primers specific to nucleic acids characteristic of (typically, genes encoding) certain AmpC beta-lactamase genes. More specifically, the present invention uses primers to identify, preferably, ampC beta-lactamase nucleic acid (typically genes), more preferably, transferable ampC beta-lactamase nucleic acid, and even more preferably, plasmid-mediated ampC beta-lactamase nucleic acid, in samples, particularly in clinical isolates of Gram-negative bacteria. The method additionally provides a method for identifying the presence or absence of AmpC beta-lactamase gene in a clinical sample. Exemplary primers of the invention include the primer sequences set forth in SEQ ID NOs: 1-12. As used herein, a nucleic acid characteristic of an AmpC beta-lactamase gene includes a gene or a portion thereof. A “gene” as used herein is a segment or fragment of nucleic acid (e.g., a DNA molecule) involved in producing a peptide (e.g., a polypeptide and/or protein). A gene can include regions preceding (upstream) and following (downstream) a coding region (i.e., regulatory elements) as well as intervening sequences (introns, e.g., non-coding regions) between individual coding segments (exons). The term “coding region” is used broadly herein to mean a region capable of being transcribed to form an RNA. The transcribed RNA can be, but need not necessarily be, further processed to yield an mRNA. A method for identifying the presence or absence of an AmpC beta-lactamase gene in a clinical sample is provided. Preferably, the clinical sample provided is characterized as a Gram-negative bacteria with resistance to beta-lactam antibiotics, and the ampC beta-lactamase nucleic acid are of a different origin relative to a bacteria's chromosomal ampC beta-lactamase nucleic acid. The method includes, providing a clinical sample; contacting the clinical sample with at least two pairs of oligonucleotide primers specific for nucleic acid of an AmpC beta-lactamase gene, wherein one primer of each pair is complementary to at least a portion of an ampC beta-lactamase nucleic acid in the sense strand and the other primer of each pair is complementary to at least a portion of an ampC beta-lactamase nucleic acid in the antisense strand; annealing the primers to the ampC beta-lactamase nucleic acid, if present; simultaneously extending the annealed primers from a 3′ terminus of each primer to synthesize an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the ampC beta-lactamase nucleic acid, if present, serves as a template for the synthesis of an extension product for the other primer of each pair; and analyzing the sample for the presence or absence of amplified products, wherein the presence of amplified products of a size characteristic of an ampC beta-lactamase nucleic acid indicates the presence of an AmpC beta-lactamase gene in the clinical sample. Analysis of the sample may include separating the amplified products from the sample and analyzing the separated amplified products for a size characteristic of a particular type of AmpC beta lactamase gene by performing electrophoresis or by performing a high-performance liquid chromatography analysis technique known as WAVE analysis. Additionally, a method for identifying different types of ampC beta-lactamase nucleic acid in a clinical sample is also provided. Preferably, the clinical sample provided is characterized as a Gram-negative bacteria with resistance to beta-lactam antibiotics, and the ampC beta-lactamase nucleic acid are of a different origin relative to a bacteria's chromosomal ampC beta-lactamase nucleic acid. The method includes, providing a clinical sample; contacting the clinical sample with at least two pairs of oligonucleotide primers specific for nucleic acid of a particular type of AmpC beta-lactamase gene, wherein one primer of each pair is complementary to at least a portion of the ampC beta-lactamase nucleic acid in the sense strand and the other primer of each pair is complementary to at least a portion of the ampC beta-lactamase nucleic acid in the antisense strand; annealing the primers to the ampC beta-lactamase nucleic acid; simultaneously extending the annealed primers from a 3′ terminus of each primer to synthesize an extension product that is complementary to the nucleic acid strands annealed to each primer wherein each extension product after separation from the ampC beta-lactamase nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair; separating the amplified products; and analyzing the separated amplified products for a size characteristic of the particular type of AmpC beta-lactamase gene. Also, a method for identifying the presence of plasmid-mediated ampC beta-lactamase nucleic acid in a clinical sample is provided. Preferably the plasmid-mediated ampC beta-lactamase nucleic acid are of a different origin relative to a bacteria's chromosomal ampC beta-lactamase nucleic acid. The method includes, contacting the clinical sample with 2-6 pairs of oligonucleotide primers specific for nucleic acid of plasmid-mediated AmpC beta-lactamase gene, wherein one primer of each pair is complementary to at least a portion of a plasmid-mediated ampC beta-lactamase nucleic acid in the sense strand and the other primer of each pair is complementary to at least a portion of a plasmid-mediated ampC beta-lactamase nucleic acid in the antisense strand; annealing the primers to the plasmid-mediated ampC beta-lactamase nucleic acid, if present; simultaneously extending the annealed primers from a 3′ terminus of each primer to synthesize an extension product that is complementary to the nucleic acid strands annealed to each primer, wherein each extension product after separation from the plasmid-mediated ampC beta-lactamase nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair; and analyzing the sample for amplified products characteristic of a plasmid-mediated AmpC beta lactamase gene. Analyzing the sample may include separating the amplified products and analyzing the separated amplified products for a size characteristic of an AmpC beta-lactamase gene. Further, the primers may be selected from the primer sequences set forth in SEQ ID NOs: 1-12. The methods described above can employ oligonucleotide primers that may be used for identifying different types of ampC beta-lactamase nucleic acid, as well as for identifying transferable and plasmid-mediated ampC beta-lactamase nucleic acid. Other oligonucleotide primers suitable for use in the methods of the present invention include primers that are specific for AmpC beta-lactamase genes designated as MOX1-2, CMY1, 8-11, LAT1-4, CMY2-7, BIL-1, DHA 1-2, ACC-1, MIR-1, ACT-1, and/or FOX 1-5b (FOX 6, see GenBank accession number AY034848). Further, a diagnostic kit for identifying ampC beta-lactamase nucleic acid in a sample is provided. The diagnostic kit includes, at least two primer pairs capable of hybridizing to a specific type of ampC beta-lactamase nucleic acid; at least one positive control and at least one negative control; and a protocol for identification of the specific type of ampC beta-lactamase nucleic acid of interest. The kit may be used for identifying, preferably, different types of ampC beta-lactamase nucleic acid, more preferably, transferable ampC beta-lactamase nucleic acid, and even more preferably, plasmid-mediated beta-lactamase nucleic acid. The kit may further include the primer pairs individually packaged within the kit. Examples Materials and Methods Bacterial strains. Bacterial strains used as controls in this study are listed in Table 1 shown below. Strains previously identified for the expression of specific plasmid-mediated ampC genes are listed as plasmid-mediated. Strains used as controls to examine the extent of cross-hybridization for specific primers with chromosomal ampC genes are listed as chromosomal. In addition, 22 clinical isolates were evaluated for the presence of plasmid-mediated ampC genes in this study. These isolates included 18 isolates of E. coli (7 of which are known controls and are listed in Table 1), 8 of K. pneumoniae, 2 of P. mirabilis, and one of E. aerogenes. Preparation of template DNA. The organisms were inoculated into 5 milliliters (ml) of Luria-Bertani broth (Difco, Detroit, Mich.) and incubated for 20 hours at 37 degrees Celsius (° C.) with shaking. Cells from 1.5 ml of an overnight culture were harvested by centrifugation at 17,310×g for 5 minutes. After the supernatant was decanted, the pellet was resuspended in 500 microliters (μl) of distilled water. The cells were lysed by heating at 95° C. for 10 minutes, and cellular debris was removed by centrifugation at 17,310×g for 5 minutes. The supernatant (1/250th volume) was used as a source of template for amplification. PCR protocol. PCR was performed in a final volume of 50 μl in 0.5 ml thin-walled tubes. Each reaction contained: 20 mM TRIS-HCL (pH 8.4), 50 mM KCl, 0.2 mM of each deoxynucleoside triphosphate, 1.5 mM magnesium chloride (MgCl2), 0.6 micromolar (μM) of primers MOXMF, MOXMR, CITMF, CITMR, DHAMF and DHAMR, 0.5 μM of primers ACCMF, ACCMR, EBCMF, and EBCMR, 0.4 μM of primers FOXMF and FOXMR, and 1.25 units of Taq DNA polymerase (Life Technologies, Rockville, Md.). Template DNA (2 μl) was added to 48 μl of master mix and then overlaid with mineral oil. The PCR program consisted of an initial denaturation step at 94° C. for 3 minutes, followed by 25 cycles of DNA denaturation at 94° C. for 30 seconds, primer annealing at 64° C. for 30 seconds, and primer extension at 72° C. for 1 minute. After the last cycle a final extension step at 72° C. for 7 minutes was added. Five microliter aliquots of PCR product were analyzed by agarose gel electrophoresis using 2% agarose (BioRad, Hercules, Calif.). Gels were stained with ethidium bromide at 10 microgram per milliliter (μg/ml) and visualized by UV ransillumination. WAVE. The Wave Nucleic Acid Fragment Analysis System (Transgenomics, Inc., Omaha, Nebr.) was used to reduce the total time required for separation and visualization of the PCR products, as compared with using gel electrophoresis. The WAVE technology uses a matched ion polynucleotide chromatography process using separation media having a non-polar surface, wherein the process uses a counterion agent and an organic solvent to release polynucleotides from the separation media (U.S. Pat. No. 6,210,885). The WAVE systems are equipped with computer controlled ovens which enclose the columns and column inlet areas (U.S. Pat. No. 6,210,885) and utilize proprietary WAVEMAKER software (Transgenomics, Inc., Omaha, Nebr.). A comparison of the methods of gel electrophoresis and WAVE technology (FIGS. 6A and 6B) was performed using ampC multiplex PCR products from representative members of each ampC family. WAVE analysis was performed using WAVE system Model Number 2100 and WAVEmaker 4.1 software (Transgenomics, Inc., Omaha, Nebr.). Samples are taken automatically by an autosampler in the WAVE system using parameters set by the operator prior to sampling. Results Dendrogram and primer design. The genes encoding AmpC beta-lactamases are of chromosomal origin, derived from members of the family Enterobacteraceae. To date, twenty-nine different AmpC beta-lactamases have been identified (FIG. 1). They can be grouped based on their chromosomal origin. For example, the genes encoding the AmpC beta-lactamases LAT-1, CMY-2 and BIL-1 are 90.4% similar to chromosomal ampC gene of Citrobacterfreundii strain OS60. The ability to group different ampC genes allows evaluation of similarity clusters. A high degree of similarity within these clusters can result in the design of primers capable of amplifying family-specific genes. Thirty sequences of different ampC genes were downloaded from the GenBank database and percent similarities analyzed using DNAsis 2.6 program (Hitachi Software Engineering Co. Ltd., Yokohama, Japan) (FIG. 1). Six different groups were identified based on percent sequence similarity. These groups include ACC (origin Hafnia alvei), FOX (origin unknown), MOX (origin unknown), DHA (origin Morganella morganii), CIT (origin Citrobacterfreundii) and EBC (origin Enterobacter cloacae). Sequences of each cluster were aligned with the CLUSTAL W multiple alignment option in the MacVector 6.5 program (Accelrys (formerly Oxford Molecular Ltd.), Princeton, N.J.) set at default parameters, and aligned sequences were used as a reference for primer design. The resulting primers were compared with all members of the different clusters in order to avoid cross hybridization. In addition, primers were evaluated for individual melting temperatures (Tm) and length. Variation between the individual primers was a Tm of 0.5° C. and a length of 2 nucleotides. The theoretical formation of primer dimers was also evaluated and found insignificant. The twelve primers designed for multiplex PCR are listed in Table 3. Individual primers were evaluated, using template DNA from the same representative members listed above, to assure that one primer set amplified only one amplicon. Amplification was only observed when each set of family-specific primers was used with template DNA from that particular ampC family. Using these parameters, only one amplicon of the predicted size was observed for each template, primer pair tested. Plasmid-mediated ampC harboring control strains analysis. Sequences of ampC genes from the same family show slight variations, resulting in the individual family member. For example sequences of members of the proposed Citrobacter-origin family have a group homology of 98.6% (FIG. 1). In order to demonstrate that sequence variation of individual family members did not influence the outcome of multiplex ampC PCR, different members of each family were used as template (FIG. 3). Amplification of products for each family member of a particular set resulted in a single amplicon of the predicted size. For example, every template of the CIT family resulted in an amplicon of 462 base pairs (FIG. 3).   Chromosomal ampC harboring control strains analysis. Due to the mobility of ampC beta-lactamases, any technique aimed to detect these genes requires its use in different genetic backgrounds, including organisms with chromosomal ampC genes, such as E. cloacae and C. freundii. Because ampC genes originated from chromosomal genes, the ampC multiplex PCR was tested for the possibility of cross-hybridization with chromosomal beta-lactamase genes of different origin. Multiplex PCR was conducted on the Chromosomal organisms listed in Table 1. No amplification was observed using DNA template from K. pneumoniae, E. coli, P. aeruginosa, S. marcescens, P. mirabilis, or E. aerogenes (FIG. 4). As expected, amplification products of the expected size for Enterobacter-origin ampC genes were obtained when DNA from E. cloacae were used as template; this represents the EBC-product of 302 base pairs (Table 3), but no other set of ampC primers cross-reacted with this chromosomal DNA. In addition, products of the expected sizes for Citrobacter-, Morganella- and Hafnia-origin ampC genes was observed when DNA from C. freundii, M. morganii, and H. alvei were used as template. In addition, DNA template prepared from a Citrobacter spp. other than C. freundii did not result in an amplified product, indicating the specificity of this primer pair. Analysis of clinical isolates. The data presented in FIGS. 2-4 indicate the specificity of the ampC multiplex PCR using highly characterized (both phenotypically and molecularly) strains. Twenty two clinical isolates were tested in order to substantiate that ampC multiplex PCR would be able to identify family-specific ampC genes also in isolates not previously characterized molecularly. Based on phenotypic characterization, these isolates were predicted to express an AmpC beta-lactamase. DNA from these isolates served as template in an ampC multiplex PCR assay (FIGS. 5A and 5B). Two PCR reactions using 2 templates (ACT-1 and FOX-1) or 4 templates (MOX-1, LAT-1, DHA-1 and ACC) were performed and separated in the same gel as markers for individual unknown reactions. PCR analysis indicated no amplification from DNA template of 12 isolates (FIG. 5A, lanes 1, 2, 3, 4, 6, 7, 8, 11 and 12; FIG. 5B, lanes 1, 2 and 11). A single product was amplified for 11 isolates. Five isolates resulted in an amplicon of approximately 200 base pairs (FOX-like) (FIG. 5A, lanes 5 and 9; FIG. 5B, lanes 3, 4 and 8), 2 isolates resulted in an amplicon of about 300 base pairs (Enterobacter-like) (FIG. 5B, lanes 7 and 9), one isolate resulted in an amplicon of about 400 base pairs (DHA-like) (FIG. 5B, lane 6), and 3 isolates generated an amplicon with a size of about 460 base pairs (Citrobacter-like) (FIG. 5A, lane 10; FIG. 5B, lanes S and 10). Template combinations of 2 or 4 templates were used as markers at the right of both gels in FIG. 5. Substantiation that the unknown isolates with specific amplified product were as predicted, one isolate was used for sequence analysis. The CIT-like result of ampC multiplex PCR (FIG. 5A, lane 10) was confirmed by the sequencing analysis, which showed a 100% identity between the base pairs of the PCR product sequenced and the gene blacCMY-2. WAVE analysis. Specificity and sensitivity are criteria used to evaluate diagnostic techniques used for identification. In clinical laboratories speed is also an important parameter. The time required to prepare template DNA and perform multiplex PCR was a total of about 1.5 hours. However, visualization of the PCR products by gel electrophoresis requires approximately four hours for high resolution of bands in 2% agarose, staining, destaining and finally interpretation of data. In order to reduce the total required time without loosing specificity and sensitivity, an HPLC-based nucleic acid analyzing technology known as WAVE was used. A comparison of gel electrophoresis and WAVE technology was performed using ampC multiplex PCR products from a representative member of each gene family (FIG. 2). Amplified products visualized by gel electrophoresis in FIG. 2. MOX (520 base pairs), CIT (462 base pairs), DHA (405 base pairs), ACC (346 base pairs), EBC (302 base pairs) and FOX (190 base pairs) correlate with the peaks observed in FIG. 6A, with retention times of 6.07 minutes, 5.78 minutes, 5.19 minutes, 4.76 minutes, 4.39 minutes, and 3.41 minutes, respectively. The initial peak at 0.5 minute and the final peak at 10 minutes in FIG. 6A correspond to injection and washing peaks, respectively.   Multiple templates of 2 (FOX and EBC), 4 (MOX, CIT, DHA and ACC), or 6 (a combination of the 2 and 4 templates) were mixed and then amplified using ampC multiplex PCR. Amplification of two and four templates resulted in amplicons of the expected sizes and was visualized by agarose gel electrophoresis and ethidium bromide staining, as shown in FIG. 6 B. However, visualization of six amplified products was difficult, resulting in only four amplified products being readily visible. A sample from the same PCR reaction which generated the six amplification products analyzed by gel electrophoresis was analyzed by the WAVE. All six products were observed by well-defined peaks (FIG. 6A). Each peak had a retention time equivalent to the observed retention times for single-template amplification, and was consistent with the expected size when compared to the size standard. The complete disclosures of all patents, patent applications, publications, and nucleic acid and protein database entries, including for example GenBank accession numbers and EMBL accession numbers, that are cited herein are hereby incorporated by reference as if individually incorporated. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention, and it should be understood that this invention is not to be unduly limited to the illustrative embodiments set forth herein.