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Methodology for the use of Green Fluorescent Protein expressing pathogens
The pathogens used in this study will be screened by direct examination for green fluorescent colonies when exposed to ultraviolet light (266 nm). The reason that a Green Fluorescent Protein detection system is used is because soil and manure have extremely varied and diverse microbial populations. Selective media for these pathogens is not suitable because it does not enumerate injured microbial cells which can be a large proportion of microorganisms when subjected to harsh environments such as ultraviolet light, drying, acidity and competing microbial populations. Utilizing the GFP detection system, nonselective media is used and both injured and healthy inoculated pathogens are enumerated to accurately assess the surviving pathogens present. Green Fluorescent Protein originates from squid (Aequorea victoria ) and does not exist in bacteria normally found in manure or soil. The GFP gene has been cloned into plasmids that are capable of replicating and producing green fluorescent protein in both Gram-negative and Gram-positive organisms. The plasmid encoding for GFP will be introduced into the selected pathogens using electroporation.
Aim 4: Development of Methods to detect pathogens in pre-harvest environments and monitor rates of development and transfer of resistance to antibiotics.
1. Methods for detection of bacterial pathogens (Campylobacter jejuni) in environmental samples.
Experiment 1. Modify existing basic TaqMan and Allelic Discrimination Assays for direct use on environmental samples.
1). Rationale: The rapid assays will be more rapid and useful if they can detect the pathogen directly in food, fecal, and other environmental samples.
2) Experimental design: The main goal is to remove contaminating materials from samples that could interfere with the PCR assays and at the same time to concentrate the bacterial cells to increase sensitivity.
a) We are currently testing immuno-magnetic beads (Dynabeads pan mouse IgG) coated with C. jejuni-specific monoclonal antibodies (Biogenesis) to help remove contaminants and to concentrate cells.
b) We would like to test diatomaceous earth which apparently is being used successfully by the USDA to achieve the same purpose.
c) After this method has been developed, the accuracy will be determined by other scientists in this group. Scientists in Nebraska, Alabama, New York and Kentucky will use the new methods to detect Campylobacter in pre-harvest and food processing environments.
Experiment 2. Perform microbiological analysis in a collaborative project funded by USDA entitled: “Risk factors for Salmonella and Campylobacter and drug resistance in dairy cattle” (PI, John Kaneene).
1). Rationale: Fluoroquinolones were approved for use in beef cattle in 1998/1999 but not for use in dairy cows. We will be receiving many CipR isolates from animals and humans from NARMS looking for the mechanisms of CipR in these field isolates. The dairy herds will not have been exposed directly to fluoroquinolones so it will be very interesting to determine if resistance exists in this population and if so at what level. The main focus is to measure the prevalence of Campylobacter spp. (Dr. Walker), Campylobacter jejuni (Linz and Mansfield), and resistance to 10 different antibiotics (Dr. Walker) in the dairy herd isolates.
2) Experimental design: Up to 24,000 bacterial isolates will be collected during a 3 year grant. We expect that about 12,000 Campylobacter isolates will be identified by selective plating. Additionally isolates collected in pre and post harvest environments in Nebraska, Alabama, Kentucky and New York will be sent to the University of Minnesota and analyzed.
3) Methods: We will run the TaqMan assay on these isolates to identify those which are C. jejuni. Isolates which are determined to be CipR by Dr. Walker (via NCCLS microbroth dilution method) will be subjected to the Allelic Discrimination assay to determine those which are CipR due to a codon 86 mutation in gyrA
Experiment 3. Mechanism of CipR in C. jejuni. Screen 50 CipR and 50 Cip susceptible (CipS; MIC 1 ug/ml) human and animal C. jejuni isolates plus 50 CipR isolates generated in the laboratory using a rapid polymerase chain reaction (PCR) TaqMan Allelic Discrimination Assay to determine if a codon 86 mutation in the QRDR of gyrA is necessary for development of CipR. Sequence the QRDR in representative isolates to confirm the rapid assay results.
1). Rationale: NARMS and Smith et al. (1999) report a high frequency of CipR (MIC 4 g/ml) in C. jejuni isolates in humans (13%) and poultry (20 %). All CipR C. jejuni human isolates collected by NARMS in 1997 and 1998 displayed a HLCR phenotype (MIC 16 ug/ml) suggesting that strains develop or acquire high level resistance rapidly and that strains with intermediate levels of resistance (between 2 and 4 g/ml) are rare. Published data from 30 C. jejuni clinical and laboratory isolates and 11 HLCR isolates generated in our laboratory suggest that a single base transition at codon 86 in the QRDR region of gyrA in C. jejuni is associated with development of HLCR. .
2). Experimental design: C. jejuni human and animal isolates collected at the collaborating states will be separated into 2 groups based on MIC: 50 CipR isolates (MIC 4 ug/ml) and 50 CipS isolates (MIC 1 ug/ml). In addition, we will generate 5 CipR isolates in the laboratory from 10 different C. jejuni CipS strains (for a total of 50 CipR laboratory isolates). All isolates will be subjected to the Allelic Discrimination Assay to identify those containing a specific C to T transition in codon 86. The QRDR from gyrA in 10 isolates from each of the 3 groups (CipR, CipS, and CipR laboratory isolates) and all CipR isolates which test negative for the C to T transition will be amplified by PCR and the nucleotide sequence determined to confirm the accuracy of the Allelic Discrimination assay and to find all mutants (including codon 86 mutants) which may not carry the C to T transition. A nucleotide sequence database for the QRDR region will be generated which measures the frequency of QRDR mutations, the specific location, and the relationship to the level of FQ resistance.
a). Strain collection; The Campylobacter isolate collection is described in Section B.
b). MIC Determination . The microbroth dilution assay is described in Section B.
c). Amplification and nucleotide sequencing of QRDR of gyrA. The PCR primers of Husmann et al. (1991) amplify a 400 nucleotide region containing the entire QRDR. Automated nucleotide sequencing will be conducted at the MSU sequencing facility in collaboration with Dr. Tom Newman. Analysis and alignment of nucleotide sequence data will be conducted using PILEUP (GCG) available on campus.
d) TaqMan Allelic Disrimination Assay. This assay will be conducted in collaboration with Dr. Tom Newman.
4). Follow up studies: If a single-step mutation in codon 86 is a primary mechanism for development of CipR, we will measure the cross-resistance of these isolates to the more effective new generation fluoroquinolone antibiotics (gatifloxacin, trovofloxacin, moxifloxacin). Structural differences in the antibiotic molecules may result in susceptibility in these Cip resistant isolates due to decreased affinity to DNA gyrase and/or affinity to multiple targets (gyrA, gyrB, parC, parE). In either case, multiple mutations would have to occur to acquire HFQR reducing the likelihood and rate of acquisition of resistance in the field.
Experiment 4. Transfer of CipR in C. jejuni Transform 10 CipS human and animal C. jejuni isolates with the QRDR of gyrA from CipR isolates and the QRDR of gyrA from CipR isolates derived from susceptible parent strains to determine if a codon 86 mutation is sufficient for development of CipR. Transform the same CipS strains with the QRDR from 10 CipS C. jejuni isolates containing a wild type codon 86 (control).
1) Rationale. If a single base mutation at codon 86 in the QRDR of gyrA is sufficient to confer the CipR phenotype, transformation of a DNA fragment containing this mutation into an isolate with a CipS genetic background (wild type gyrA, gyrB, parC, and parE) should confer CipR.
2) Experimental design. 10 CipS human and animal strains will be transformed with 3 different DNA fragments: a) the QRDR from a single CipR isolate generated in the laboratory from each of 10 CipS C. jejuni recipient strains; b) the QRDR from the 10 CipS recipient strains (controls); and the QRDR from 10 CipR human and animal isolates. The nucleotide sequence for each QRDR fragment will be determined as part of Experiment 1 to demonstrate that the codon 86 mutation is the only base change in this QRDR fragment.
a) PCR amplification of the QRDR and nucleotide sequence analysis. Same as for Specific Aim 1
b) Natural transformation. Recipients strains will be transformed with 0.5 to 1 ug of the appropriate QRDR fragment by natural transformation and selected for ability to grow on Brucella Agar with Sheep’s Blood (BASB) containing Cip at 4 g/ml. Transformants will be retested on BASB with Cip at 16g/ml to determine the number of CipR transformants that are HLCR.
Experiment 5. Mechanism of ErmR in C. jejuni. Screen 50 ErmR and 50 Erm susceptible (ErmS; MIC 0.5 g/ml) C. jejuni isolates by PCR and reverse transcriptase (RT) PCR analyses to measure the relative contribution of 2 potential mechanisms for development of ErmR: a) specific mutations in the 23S rRNA, and b) the expression of ermF.
1). Rationale: We recently identified an erm homolog in C. jejuni with 29% identity and 42% similarity overall to ermF from Bacteroides fragilis (Rasmussen et al.,1986) and localized identities to other erm genes. Because ErmR isolates of C. jejuni studied to date are cross-resistant to lincosamide and streptogramin B (Taylor, 1992), this suggests that a MLS resistance mechanism conferred by our ermF homologue is a distinct possibility. Based on these new data, we proposed that transfer or activation of an ermF homologue is one primary mechanism of conferring the ErmR phenotype in C. coli and C jejuni.
2). Experimental design: Using a PCR assay and appropriate primers, we will screen 50 ErmR and 50 ErmS isolates for the relative proportion which are resistant via each of the two potential resistance mechanisms discussed above (mutations in 23S; RNA di-methylase). First, we will amplify the ermF homologue from genomic DNA of an ErmR isolate using appropriate PCR primers. We will analyze each of the ErmR and ErmS isolates using these same PCR primers to detect the presence of ermF and utilize the cloned gene to transform Erm susceptible C. jejuni and C. coli isolates. If transformation confers resistance and the gene is present in resistant and susceptible isolates, we will determine the nucleotide sequence of both genes to identify a mutation that may result in gene activation by a change in activity or expression levels. Reverse transcriptase PCR (RT- PCR) of resistant and susceptible isolates will be used to measure transcript levels for the Erm homologue to determine if the gene, if present, is expressed in all isolates. Second, we will use appropriate primers to amplify Domain V of the 23S rRNA gene in the ErmR and ErmS isolates to determine the frequency of mutations in this domain. Genomic DNA and cloned 23S DNA fragments will be used to transform susceptible isolates to confirm that the mutations detected in the PCR screening assay are sufficient to confer ErmR.
a) PCR cloning/ PCR screening assay. We will use standard procedures outlined in Current Protocols in Molecular Biology; Ausubel et al. (1999; Chapter 15). (ii) RT PCR of Erm transcript. We will use the procedure of Pickens et al. (1999) for amplification of the erm transcript.
b) Transformation of C. jejuni and C. coli. Transformation will be performed using the published (Wang and Taylor, 1990; Wassenaar et al., 1993) natural transformation procedures already established in our laboratory.
4). Follow up studies. If the ermF homologue confers resistance, we will study the rate of intra-strain, inter strain and inter species transfer of this gene.
Objective 2. Chemical and Physical Decontamination in Food Processing Plant Environments
Chemical treatment to reduce the load of spoilage and pathogenic
microorganisms on raw food commodities has been extensively researched.
While efficacy has been clearly demonstrated in many laboratory
experiments, few novel chemical treatments have been adopted into
commercial processing of raw food commodities. The lack of commercial
use of such treatments, in a time when food safety has emerged as the
key consumer issue, serves to demonstrate the need for further work in
this area. Processors have been hesitant to adopt new technologies, in
part, due to a lack of consistent data. That is, data often vary from
one study to the other. These inconsistencies in results are primarily
due to difference in experimental procedures and conditions. There is a
great variety of methods that have been used by researchers when
investigation potential antimicrobial treatments. To compare results for
one study to another, methodologies for testing potential treatments for
raw commodities must be more standard. Use of a more standard method of
testing will allow processors to confidently adopt procedures as part of
their pathogen control systems. Furthermore, validation of procedures
for reducing pathogens will be facilitated by a better understanding of
the influence of experimental variables on antimicrobial efficacy."
Aim 1. Develop Method for Determining the Efficacy of Pathogen Reduction (Decontamination)Treatments for Raw Food Commodities
Within the participants of this project, there is a great deal of experience in investigating antimicrobial treatments. Therefore, these participants, who have used a wide variety of assessment methods, will undertake a commitment to developing a central method and recommendation for researchers to use when quantifying antimicrobial activity of novel decontaminants.
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