Progress 01/29/16 to 06/30/18
Outputs
Target Audience:There are several target audiences for this project. First and possibly most important is the poultry productionindustry. Findings from this study will be translated for this end-user audience so that the results can be applied to mitigateFQR Campylobacter in the poultry production system. A second target audience is the scientific community, mainlymicrobiologists and epidemiologists. Finally, we anticipate that government agencies, such as FDA, USDA and CDC, will usethe results of this project to better understand Campylobacter ecology and the relationship between animal production andhuman illness. Changes/Problems:We usedseveral alternative approaches to express the microcin in the E. coli strain. We have confirmed the presence ofthe gene in the E. coli strain. We are also beginning the iron experiments. What opportunities for training and professional development has the project provided?A PhD student was trained on this grant. How have the results been disseminated to communities of interest?A poster was presented at the 3rd International Conference on One Medicine One Science (iCOMOS) which took place on April 30, 2018 in Minneapolis, MN. Peer-reviewed manuscripts are being prepared. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The first specific aim hypothesizes that the point mutation in gyrA responsible for FQ resistance in C. jejuni enhances iron acquisition in regulation which gives fitness benefits to FQR C. jejuni in poultry intestine where bioavailability of iron is limited. Five FQS C. jejuni (ciprofloxacin MIC ≤ 1µg/mL) underwent antibiotic selections on Mueller-Hinton agar to create complementary FQR C. jejuni strains (ciprofloxacin MIC ranging between 16 - 64µg/mL). gyrA was sequenced using Sanger sequencing approach to confirm the point mutation for FQR. The first specific aim hypothesizes that the limited bioavailability of iron in broiler chickens enhances fitness and persistence of FQ-R C. jejuni over FQ-S C. jejuni. Previous research showed that FQ-R C. jejuni had significantly increased expression of iron acquisition and regulation genes in vitro compared to FQ-S C. jejuni. Taking the previous research further, we proposed to conduct experiments to measure and compare expressions of the genes that were significantly upregulated in the previous study in iron-depleted conditions over time. Five FQS C. jejuni (ciprofloxacin MIC ≤ 1µg/mL) underwent antibiotic selections on Mueller-Hinton agar to create complementary FQR C. jejuni strains (ciprofloxacin MIC ranging between 16 - 64µg/mL). gyrA was sequenced using Sanger sequencing to confirm the point mutation for FQR C .jejuni. gyrB was sequenced to confirm the presence of any additional point mutations caused by the acquisition of FQ resistance in FQR C. jejuni. The sequencing results showed that all FQR C. jejuni strains acquired a nonsynonymous mutation in gyrA at position 86 which changed threonine to isoleucine. Two of the FQR C. jejuni showed an additional point mutation in the gyrB gene at position 460 which changed serine to phenylalanine. The potential effect of this additional point mutation in gyrB gene has not been investigated. The growth kinetics of FQS and FQR C. jejuni were compared by running growth curves experiments thrice with three technical replicates. Optical density at 600 (OD600) measured at 0, 6, 24, 30 and 48 hours was compared between FQS and FQR C. jejuni strains, and no statistical differences were observed within pairs. To induce iron-depleted condition, 2,2'-dipyridyl, an iron chelating agent was added at 0μM or 100μM concentration to FQ-R and FQ-S C. jejuni broth cultures in mid-log phase. The original protocol included 200μM concentration but at this concentration Campylobacter culture did not grow well, and as the result extracted RNA was of poor quality and low yield. Once 2,2'-dipyridyl was added, 1mL of culture was collected at 0, 3, 5, 10, 30 and 60 minutes. RNA was extracted from each culture using TRIzol extraction method, treated with DNase and RNAse inhibitor, standardized using Nuclease-free water (at 10ng/μL), and finally reverse transcribed to cDNA. We tested three reference genes (slyD and two different primer sets of rpoA) and chose rpoA as a reference gene. qPCR was run to compare relative gene expressions of six genes (Cj0179, Cj1661, Cj0426, Cj0427, Cj0334 and Cj1664) between FQR and FQS C. jejuni using rpoA gene as a reference gene as suggested by Ritz et al. (2009). The results of the qPCR showed that relative expressions of the tested genes were not statistically different (using Pfaffl delta-delta approach and REST 2009 bootstrapping approach) between FQR and FQS C. jejuni across time points. Only one pair showed consistent upregulation in FQR C. jejuni compared to FQS C. jejuni that was statistically significant. The FQR C. jejuni of this pair had additional point mutation in gyrB gene. Additionally, we conducted experiments to measure tolerance to oxidative stress in FQR and FQS C. jejuni in vitro. Three µL of 3% hydrogen peroxide or two-fold diluted 3% hydrogen peroxide in water (1/2, 1/4, and 1/8 dilutions) was added to a disk and placed on the top agar layer containing lawn of FQR or FQS C. jejuni and incubated for 24 hours. The results showed that both FQR and FQS C. jejuni were susceptible to different dilutions of 3% hydrogen peroxide. No differences in the inhibition sizes was observed between FQR and FQS C. jejuni. The second specific aim hypothesizes that the presence and activity of mccB17 produced by poultry commensal E. coli as another indirect selective pressure favoring persistence of FQR C. jejuni in broiler chickens. Our previous work showed that when E. coli cell lysates containing active microcin B17 was applied to top agar layer containing FQ-R or FQ-S C. jejuni, clear inhibition zones that were similar in sizes were observed, indicating that microcin B17 inhibits growth of Campylobacter regardless of fluoroquinolone resistance. However, previous studies in E. coli showed that the mode of inhibition of microcin B17 is similar to that of fluoroquinolone-class of antibiotics, and as such, FQ-R E. coli was resistant to microcin B17 until a certain concentration was achieved. Thus, it is possible that FQ-R and FQ-S C. jejuni may react differently to different concentrations of microcin B17. To test this hypothesis, E. coli DH5α harboring a pUC19 plasmid containing mcb operon which is responsible for production and exportation of mccB17 was grown in M63 minimal media with ampicillin (100µg/mL) for 40 hours. Cell pellets were lysed by boiling in 100mM acetic acid and 1mM EDTA for 10 minutes. Cell extract was collected and filtered through 0.2mM membrane filter. Spot-plate assays were conducted to test the effects of cell extract containing active mccB17 on growth of FQR and FQS C. jejuni strains. Top agar layers (0.7% agar) containing E. coli DH5α (positive control), E. coli DH5α harboring a pUC19 with mcb operon (negative control) and FQR and FQS C. jejuni were prepared. Five µL of the cell lysate was directly applied to the top agar layer. After 24 hours of incubation, zone of inhibition was observed. This experiment showed that regardless of FQ resistance phenotype, both FQR and FQS C. jejuni strains were susceptible to the activity of mccB17. The cell lysate was two-fold diluted in water (1/2, 1/4, 1/8, 1/16 and 1/32) to understand the relative concentration at which the growth of FQR and FQS C. jejuni was sustained in the presence of mccB17. The result of this experiment showed that while there were differences among strains, all strains exhibited no growth between 1/4th and 1/16th dilution. Additionally, a total of 391 E. coli isolates recovered from boot sock samples collected from broiler farms in U.S. was screened for the presence of 5.1kb mcb operon. Out of 391 isolates screened, 1 isolate was confirmed by PCR to contain the mcb operon. However, when cell lysate was obtained from this isolate and spot-tested on E. coli DH5α to check for the production of active mccB17, no zone of inhibition was observed.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
A poster was presented at the 3rd International Conference on One Medicine One Science (iCOMOS) which took place on April 30, 2018 in Minneapolis, MN.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:There are several target audiences for this project. First and possibly most important is the poultry production industry.Findingsfrom this study will be translated for this end-user audience so that the results can be applied to mitigate FQRCampylobacter inthe poultry production system. A second target audience is the scientific community, mainly microbiologists andepidemiologists.Finally, we anticipate that government agencies, such as FDA, USDA and CDC, will use the results of this project to betterunderstand Campylobacter ecology and the relationship between animal production and human illness. Changes/Problems:We are using several alternative approaches to express the microcin in the E. coli strain. The inability to express the microcin has delayed this project. What opportunities for training and professional development has the project provided?A PhD student is being trained on this grant. How have the results been disseminated to communities of interest?An abstract will be submitted for a poster presentation at the 3rd International Conference on One Medicine One Science (iCOMOS) which will be held on May, 2018. What do you plan to do during the next reporting period to accomplish the goals?We are using several alternative approaches to express the microcin in the E. coli strain. We have confirmed the presence of the gene in the E. coli strain. We are also beginning the iron experiments. By the next reporting period, this project and its goals will likely be completed.
Impacts
What was accomplished under these goals?
The first specific aim hypothesizes that the limited bioavailability of iron in broiler chicken enhances fitness and persistence of FQ-R C. jejuni over FQ-S C. jejuni. Previous research showed that FQ-R C. jejuni had significantly increased expression of iron acquisition and regulation genes in vitro compared to FQ-S C. jejuni. Taking the previous research further, we proposed to conduct experiments to measure and compare expressions of the genes that were significantly upregulated in the previous study in iron-depleted conditions over time. To induce iron-depleted condition, 2,2'-dipyridyl, an iron chelating agent was added at 0µM or 100µM concentration to FQ-R and FQ-S C. jejuni broth cultures in mid-log phase. The original protocol included 200µM concentration but at this concentration Campylobacter culture did not grow well, and as the result extracted RNA was of poor quality and low yield. Once 2,2'-dipyridyl was added, 1mL of culture was collected at 0, 3, 5, 10, 30 and 60 minutes. RNA was extracted from each culture using TRIzol extraction method, treated with DNase and RNAse inhibitor, standardized using Nuclease-free water (at 10ng/µL), and finally reverse transcribed to cDNA. We tested three reference genes (slyD and two different primer sets of rpoA) and chose rpoA as a reference gene. We are currently running qPCR on six genes (Cj0179, Cj1661, Cj0426, Cj0427, Cj0334, and Cj1664) and hope to finish this portion of the project by end of January. Additionally, we conducted experiments to measure tolerance to oxidative stress in FQ-R and FQ-S C. jejuni in vitro. Three µL of 3% hydrogen peroxide or two-fold diluted 3% hydrogen peroxide in water (1/2, 1/4 and 1/8 dilutions) was added to a disk and placed on the top agar layer of Campylobacter and incubated for 48hours. The results showed that both FQ-R and FQ-S C. jejuni were susceptible to different dilutions of 3% hydrogen peroxide. No differences in the inhibition sizes was observed between FQ-R and FQ-S C. jejuni. The second specific aim hypothesizes that the presence and activity of microcin B17 produced by poultry commensal Escherichia coli (E. coli) as another indirect selective pressure favoring persistence of FQ-R C. jejuni in broiler chickens. Our previous work showed that when E. coli cell lysates containing active microcin B17 was applied to top agar layer containing FQ-R or FQ-S C. jejuni, clear inhibition zones that were similar in sizes were observed, indicating that microcin B17 inhibits growth of Campylobacter regardless of fluoroquinolone resistance. However, previous studies in E. coli showed that the mode of inhibition of microcin B17 is similar to that of fluoroquinolone-class of antibiotics, and as such, FQ-R E. coli was resistant to microcin B17 until a certain concentration was achieved. Thus, it is possible that FQ-R and FQ-S C. jejuni may react differently to different concentrations of microcin B17. Multiple dilutions of cell lysates containing active microcin B17 has been prepared (two-fold diluted in water). These diluted cell lysates will be used in disk inhibition assays and growth curves experiments to better understand how various concentrations of microcin B17 affect fitness and growth of FQ-R and FQ-S C. jejuni. Additionally, about 400 E. coli isolates obtained from broiler farms were screened to measure the prevalence of occurrence of microcin B17 producing E. coli in broiler chickens. One isolate contained mcb operon confirmed by PCR.
Publications
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Progress 01/29/16 to 09/30/16
Outputs
Target Audience:There are several target audiences for this project. First and possibly most important is the poultry production industry. Findings from this study will be translated for this end-user audience so that the results can be applied to mitigate FQR Campylobacter in the poultry production system. A second target audience is the scientific community, mainly microbiologists and epidemiologists. Finally, we anticipate that government agencies, such as FDA, USDA and CDC, will use the results of this project to better understand Campylobacter ecology and the relationship between animal production and human illness. Changes/Problems:We are using several alternative approaches to express the microcin in the E. coli strain. The inability to express the microcin has delayed this project. What opportunities for training and professional development has the project provided?A PhD student is being trained on this grant. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are using several alternative approaches to express the microcin in the E. coli strain. We have confirmed the presence of the gene in the E. coli strain. We are also beginning the iron experiments.
Impacts
What was accomplished under these goals?
A major goal of this project is to understand the emergence and persistence of fluoroquinolone-resistant (FQ-R) Campylobacter jejuni (C. jejuni) in broiler chickens. Previous research showed that FQ-R Campylobacter can outcompete fluoroquinolone-susceptible (FQ-S) Campylobacter both in vitro and in vivo. Because broiler chicken is considered to be the major source of campylobacteriosis in humans and the fluoroquinolone antibiotic class is one of the treatment options for campylobacteriosis, it is imperative to understand how FQ-R Campylobacter emerges and persists in broiler chickens despite the ban of fluoroquinolone use in the poultry industry in U.S. The overall hypothesis of the project is that two key factors, limited bioavailability of iron in broiler chickens and the activity of a specific microcin produced by poultry commensal Escherichia coli (E. coli) are indirect selective pressures involved in the enhanced fitness and persistence of fluoroquinolone-resistant C. jejuni in broiler chickens. The project was investigated using five wild-type FQ-S (minimum inhibitory concentration < 1 µg/mL) C. jejuni strains isolated from five different broiler farms and five FQ-R complementary C. jejuni strains created by antibiotic selection using ciprofloxacin (4 µg/mL). The minimum inhibitory concentration (MIC) of the FQ-R complementary strains ranged from 16 - 32 µg/mL. To check isogenicity between FQ-R and FQ-S strains, gyrA was sequenced to confirm the presence of a point mutation at position Thr-86, Asp-90 or Ala-70 which confers fluoroquinolone resistance in Campylobacter. Additionally, gyrB was sequenced to confirm the lack of compensatory mutations. All five FQ-R complementary strains showed the point mutation at position Thr-86 in gyrA. However, two of the FQ-R complementary strains had a nonsynonymous point mutation at position 449 in gyrB. Currently, we do not know the function of this nonsynonymous point mutation in gyrB. All ten strains are currently undergoing growth curves in Mueller-Hinton media to confirm that growth characteristics of FQ-R and FQ-S strains are comparable. The OD600 is measured at 0, 6, 24, 30 and 48 h and colony forming unit (CFU) is measured at each time point by plating two dilutions on Mueller-Hinton agar. The growth curve experiment is conducted thrice with three biological replicates (duplicate plating per replicate per dilution). To investigate the effect of microcin B17 on the fitness of C. jejuni, E. Coli ZK357 strain harboring the pPy113 plasmid that possess mcb operon which encodes for MccB17 production and export was obtained from Dr. Roberto Kolter. Commercially available M63 medium broth was used to culture E. coli ZK356 and E. coli K12 (as negative control) and cell supernatant and lysate were extracted. The zone of inhibition experiment was conducted using extracted cell supernatant and lysate on E. coli ZK356 and E. coli K12 cultures to confirm the presence and activity of microcin B17. However, the experiment failed to show that microcin B17 was produced by ZK356 strain and present in either cell supernatant or lysate. As such, polymerase-chain-reaction (PCR) was conducted to confirm the presence of mcb operon in ZK356 strain. The PCR result showed that 5.1 kb mcb operon with its internal promoter is present in ZK356 strain but absent in K12 strain, confirming that ZK356 strain can produce microcin B17. Additional work will be conducted as suggested by Fang and Demain (1997) to produce and extract microcin B17 from ZK356 strain.
Publications
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