Resistance in Soil ANAS 16 · r í ò^ ZE P v À ] ( ] ] } v > v í W J ì ì o Title: Microsoft...

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- 16S rRNA gene verification Lane 1: !00 bp ladder Lane 2: Soil StrA Lane 3: Bacterial Water StrA Lane 4: Colony Bacteria StrA Lane 5: StrA Positive Control Lane 6: 16S Positive Control Lane 7: 16S Soil Lane 8: 16S Bacteria Water Lane 9: 16S Colony Bacteria Lane 10: Negative Control 1 2 3 4 5 6 7 8 9 10 Kirby-Bauer Results Results for 16S 466 bp product The Presence of 16S rRNA was used to verify bacterial DNA in all samples including negative results. Samples from the RC pig farm (left) that produced positive results for StrA and TetM also demonstrated less sensitivity to antibiotics using the Kirby-Baur method Methods and Conclusion The methods used in this study are effective in evaluating the presence or absence of specific resistance gene sequences in samples taken in situ. Although there is not enough data to support a specific correlation between sample location and the presence of resistance genes, it is notable that the only sample to demonstrate both the presence of resistance genes and to express resistance was taken from an agricultural site. This farm does not use antibiotics in feed, but feed from other sources, and the pigs themselves may have detectable resistance genes present. Thus, future studies will be needed to explore the possible correlation between in vivo antibiotic use and the local spread of resistant bacteria. This work was support by NASA COP grant awarded to CSN. Methods PCR for Resistance Genes Lane 1 Mol Wt. Ladder 100 bp. Lane 2 Wetlands soil (tetM) Lane 3 Wetlands water (tetM) Lane 4 Negative control (tetM) Lane 5 Blank Lane 6 Unknown Pig Soil (tetM) Lane 7 Positive control (tetM) Lane 8 100 bp Mol wt ladder Lane 1. 100 bp Mol Wt. ladder Lane 2. Harriet lake soil (tetM) Lane 3. Positive control (tetM) Lane 4. Negative control (tetM) Lane 5. Red Rock soil (tetM) Lane 6. Red Rock water (tetM) Lane 1: Negative control Lane 2: 100 bp Ladder Lane 3: Pig Soil (strA) Lane 4: Pig water - (strA) Lane 5: Positive control (strA) Lane 6: Wetlands soil (strA) Lane 1: Ladder Lane 2: Soil Harriet Lake - (strA) Lane 3: Water Harriet Lake (strA) Lane 4: Soil Red Rock - (strA) Lane 5: Negative control - (strA) Lane 6: Positive control - s (strA) Lane 7: Positive control - (strA) Antibiotic Resistance Genes in Las Vegas Valley Bacteria Dhalai, A., Harger, S., Ortega, D., Sottile, R., Kimberly, K., Harbour, D. V. Abstract A recent British study concluded that annually, there are 700,000 deaths world wide that can be attributed to antibiotic resistant bacteria. [1] Mapping the spread of resistance genes in the wild is a necessity as a method to measure both the impact of antibiotic over-use on bacterial populations in situ rather than in vivo, and to determine the presence of resistance genes in wild bacterial populations that could be selected for in the presence of antibiotics even if resistance has not yet been expressed. The purpose of this study was two fold; to establish procedures that would allow for the detection of resistance genes in local soil and water samples, and to use these methods to assess samples from four discreet locations in the Las Vegas Valley. One sample produced repeatable positive PCR results for two antibiotic resistance genes. Sample Locations Clockwise from top Left, Las Vegas Wetlands Park, Lake Harriet Las Vegas, RC Farms North Las Vegas, Red Rock Canyon Conservation Area Methods ≠ Soil was plated on LB agar with amphotericin B using ten fold dilution scheme from 10 -3 -10 -8 to determine CFU/gm of Soil CFU/gm Soil and genomic DNA Correlations of baterial CFU/gm soil with concentrations of genomic DNA. Nanodrop 260:280 ratio was 1.68. Primer Alignments Graphical representation of PCR primers and their binding locations from the NCBI PrimerBLAST online tool. The 409 Bp pair shown as Primer 3 was used to screen for Str-A. The 67 Bp Primer used for TetM is not shown above. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 1 2 3 4 5 6 12 3 4 5 6 7 Results for tetM 67 bp product Results for strA 409 bp product Results detected the presence of the strA and tetM genes in pig farm soil and water but not in Red Rock, wetlands, or Lake Harriet soil and water Results detected the presence of the strA and tetM genes in pig farm soil and water but not in Red Rock, wetlands, or Lake Harriet soil and water

Transcript of Resistance in Soil ANAS 16 · r í ò^ ZE P v À ] ( ] ] } v > v í W J ì ì o Title: Microsoft...

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16S rRNA gene verification

Lane 1: !00 bp ladderLane 2: Soil StrALane 3: Bacterial Water StrALane 4: Colony Bacteria StrALane 5: StrA Positive ControlLane 6: 16S Positive Control Lane 7: 16S Soil Lane 8: 16S Bacteria WaterLane 9: 16S Colony Bacteria Lane 10: Negative Control

1 2 3 4 5 6 7 8 9 10

Kirby-Bauer Results

Results for 16S 466 bp product

The Presence of 16S rRNA was used to verify bacterial DNA in all samples including negative results.

Samples from the RC pig farm (left) that produced positive results for StrA and TetM also demonstrated less sensitivity to antibiotics using the Kirby-Baur method

Methods and Conclusion

The methods used in this study are effective in evaluating the presence or absence of specific resistance gene sequences in samples taken in situ. Although there is not enough data to support a specific correlation between sample location and the presence of resistance genes, it is notable that the only sample to demonstrate both the presence of resistance genes and to express resistance was taken from an agricultural site. This farm does not use antibiotics in feed, but feed from other sources, and the pigs themselves may have detectable resistance genes present. Thus, future studies will be needed to explore the possible correlation between in vivo antibiotic use and the local spread of resistant bacteria. This work was support by NASA COP grant awarded to CSN.

Methods

PCR for Resistance Genes

Lane 1 Mol Wt. Ladder 100 bp.Lane 2 Wetlands soil (tetM)Lane 3 Wetlands water (tetM)Lane 4 Negative control (tetM) Lane 5 BlankLane 6 Unknown Pig Soil (tetM) Lane 7 Positive control (tetM) Lane 8 100 bp Mol wt ladder

Lane 1. 100 bp Mol Wt. ladderLane 2. Harriet lake soil (tetM)Lane 3. Positive control (tetM)Lane 4. Negative control (tetM)Lane 5. Red Rock soil (tetM)Lane 6. Red Rock water (tetM)

Lane 1: Negative controlLane 2: 100 bp LadderLane 3: Pig Soil (strA)Lane 4: Pig water - (strA)Lane 5: Positive control (strA)Lane 6: Wetlands soil (strA)

Lane 1: LadderLane 2: Soil Harriet Lake - (strA)Lane 3: Water Harriet Lake (strA)Lane 4: Soil Red Rock - (strA)Lane 5: Negative control - (strA)Lane 6: Positive control - s (strA)Lane 7: Positive control - (strA)

Antibiotic Resistance Genes in Las Vegas Valley BacteriaDhalai, A., Harger, S., Ortega, D., Sottile, R., Kimberly, K., Harbour, D. V.

AbstractA recent British study concluded that annually, there are 700,000 deaths world wide that can be attributed to antibiotic resistant bacteria. [1] Mapping the spread of resistance genes in the wild is a necessity as a method to measure both the impact of antibiotic over-use on bacterial populations in situ rather than in vivo, and to determine the presence of resistance genes in wild bacterial populations that could be selected for in the presence of antibiotics even if resistance has not yet been expressed. The purpose of this study was two fold; to establish procedures that would allow for the detection of resistance genes in local soil and water samples, and to use these methods to assess samples from four discreet locations in the Las Vegas Valley. One sample produced repeatable positive PCR results for two antibiotic resistance genes.

Sample Locations

Clockwise from top Left, Las Vegas Wetlands Park, Lake Harriet Las Vegas, RC Farms North Las Vegas, Red Rock Canyon Conservation Area

Methods

≠

Soil was plated on LB agar with amphotericin B using ten fold dilution scheme from 10-3-10-8 to determine CFU/gmof Soil

CFU/gm Soil and genomic DNA

Correlations of baterial CFU/gm soil with concentrations of genomic DNA. Nanodrop 260:280 ratio was 1.68.

Primer Alignments

Graphical representation of PCR primers and their binding locations from the NCBI PrimerBLAST online tool. The 409 Bp pair shown as Primer 3 was used to screen for Str-A. The 67 Bp Primer used for TetM is not shown above.

1 2 3 4 5 6 7 8

1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4 5 6 7

Results for tetM 67 bp product

Results for strA 409 bp product

Results detected the presence of the strA and tetM genes in pig farm soil and water but not in Red Rock, wetlands, or Lake Harriet soil and water

Results detected the presence of the strA and tetM genes in pig farm soil and water but not in Red Rock, wetlands, or Lake Harriet soil and water

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