Progress 10/01/12 to 09/30/17
Outputs
Target Audience:Microbiologists and Regulatory Agencies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project constituteda part of the Ph.D. dissertation of a graduate student, and provided opportunities for training the student in detecting antibiotic and chlorine resistance in A. baumannii using both traditional and molecular techniques. The project also provided opportunities for the student to present the research findings at a national conference. 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?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Determine the prevalence of MDR A. baumannii on fresh produce in Connecticut A total of 300 samples of lettuce, carrots and potatoes (n=100) were collected from 14 farmers markets in Connecticut, and tested for the presence of antibiotic resistant bacteria, including A. baumannii by plating. Among the produce tested, 73% lettuce, 86% carrots and 74% potatoes were found to harbor bacteria resistant to multiple antibiotics. These bacteria were isolated in pure cultures and identified using API 20NE as Burkholderia cepacia/gladioli (48%), Pseudomonas luteola (41%), Stenotrophomonas maltophilia (18%) and Acinetobacter baumannii/calcoaceticus (1.3%). In addition, the antibiotic resistance profile of each of these isolates was determined using antibiotic disc diffusion assay. High resistance rates were observed for ceftriaxone (90%), streptomycin (85%) and neomycin (72%) in Burkholderia cepacia/gladioli; for imipenem (10%) and colistinsulphate (30%) in Pseudomonas luteola; for doxycycline (56%), imipenem (70%), erythromycin (80%) and minocycline (41%) in Stenotrophomonas maltophilia, and for imipenem (75%), rifampcin (25%), streptomycin and ceftriaxone (100%) in Acinetobacter baumannii/calcoaceticus. The results reveal the high prevalence of multi-drug resistant bacteria, including A. baumannii on fresh produce, thereby highlighting the potential health impact in consumers, especially those with a compromised immune system. 2.Investigate the effect of chlorine on the survival of multi-drug resistant Acinetobacter baumannii, and expression of its antibiotic resistance genes. This study investigated the effect of different levels of chlorine on the viability of A. baumannii in water. Additionally, the effect of chlorine exposure on the transcription of genes conferring antibiotic resistance in A. baumannii was studied. Eight clinical isolates of A. baumannii were separately exposed to different chlorine concentrations (0.2, 1, 2, 3, and 4 ppm) with a contact time of 30, 60, 90 and 120 sec. The surviving pathogen counts at each specified contact time were determined using broth dilution assay. In addition, the expression of A. baumannii efflux pump genes (adeA, adeB, adeC, abeM), and those encoding resistance to antibiotics, including β-lactams (blaP), aminoglycoside (ami), chloramphenicol (cmr), sulfamethoxazole (sul1), tetracycline (tetA), and multiple drug resistance protein (mdrp) was determined by real-time quantitative PCR (RT-qPCR) in three isolates following exposure to chlorine.An up-regulation of all or some of the antibiotic resistance genes in the isolates, indicating a chlorine-associated induction of antibiotic resistance in the pathogen. 3. Effect of Trans-cinnamaldehyde and Eugenol on decreasing A. baumannii resistance to β-lactam antibiotics. This study investigated the efficacy of two food-grade, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde(TC) and eugenol (EG) in decreasing A.baumannii's resistance to seven β-lactam antibiotics, including ampicillin, methicillin, meropenem, penicillin, aztreonam, amoxicillin and piperacillin. Two MDR A.baumannii isolates (ATCC 17978 and AB251847) were separately cultured in tryptic soy broth (~6logCFU/ml) containing the minimum inhibitory concentration (MIC) of TC or EG with or without the MIC of each antibiotic at 37oC for 18 h. A.baumannii strains not exposed to the PDAs or antibiotics served as controls. Following incubation, A.baumannii counts were determined by broth dilution assay. In addition, the effect of PDAs on the permeability of outer membrane and efflux pumps in A. baumannii was measured. Further, the effect of TC and EG on the expression of A.baumannii genes encoding resistance to β-lactam antibiotics (blaP), efflux pumps (adeABC) and multi-drug resistant protein (mdrp) was studied using real-time quantitative PCR (RT-qPCR). The experiment was replicated three times with duplicate samples of each treatment and control. The results from broth dilution assay indicatedthat both TC and EG in combination with antibiotics increased the sensitivity of A.baumannii to all the tested antibiotics (P<0.05). The two PDAs inhibited the function of A. baumannii efflux pump, (AdeABC), but did not increase the permeability of its outer membrane. Moreover, RT-qPCR data revealed that TC and EG down-regulated the expression of majority of the genes associated with β-lactam antibiotic resistance, especially blaPand adeABC (P<0.05).The results suggest that TC and EG could potentially be used along with β-lactam antibiotics for controlling MDR A.baumannii infections; however, their clinical significance needs to be determined using in vivo studies. 4. Efficacy oftrans-cinnamaldehyde (TC) and eugenol (EG) for controlling wound infections of A. baumannii in vitro This study investigated the efficacy of two natural, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde (TC) and eugenol (EG) for decreasing A. baumannii adhesion to and invasion of normal human keratinocytes (HEK001). The efficacy of two PDAs for inhibiting A. baumannii biofilm formation was also determined using an in vitro collagen matrix wound model. In addition, the effect of TC and EG on A. baumannii biofilm architecture was visualized using confocal scanning microscopy. Furthermore, the effect of both PDAs on A. baumannii genes critical for biofilm synthesis was determined using real-time quantitative PCR (RT-qPCR). Both TC and EG significantly reduced A. baumannii adhesion to HEK001 by ~2 to 2.5 log CFU/ml (P < 0.05), and invasion by ~2 to 3 log CFU/ml, compared to the controls (P < 0.05). Further, after 24 and 48 h, TC inhibited biofilm formation by ~1.5 and ~2 to 3.5 logCFU/ml, while EG decreased biofilm-associated bacteria by ~2 and ~3.5 log CFU/ml, respectively, compared to controls (P < 0.05). Confocal microscopy revealed that TC and EG resulted in the death of biofilm-associated A. baumannii, and disrupted the biofilm architecture. RT-qPCR results indicated that the two phytochemicals significantly down-regulated the transcription of genes associated with A. baumannii biofilm production.The results suggest that both TC and EG could potentially treat A. baumannii wound infections; however, their efficacy in in vivo models needs to be validated. 5. Development and Optimization of aReal-Time quantitative PCR for specific detection of A. baumannii in water and blood. Polymerase chain reaction is a powerful molecular technique, which has been widely used for detecting microorganisms. The objective of this study was to develop a real-time polymerase chain reaction (RT-PCR) for specifically detecting A. baumannii in water and blood using TaqMan primer/probe set targeting a highly conserved 102-bp DNA sequence in adeT, an efflux pump found in A. baumannii. For testing the limit of detection, RT-PCR was done directly with 10-fold dilutions of A. baumannii suspension in blood or water (106 CFU to 101 CFU) and various concentrations of genomic DNA. Further, the sensitivity of the RT-PCR was tested after enrichment of A. baumanniiin tryptic soy broth. The results revealed that all the A. baumanniiisolates yielded a 102-bp PCR product, however, none of the tested negative control isolates, including other Acinetobacter species produced any amplification. The sensitivity of PCR for detecting A. baumannii in blood and water was 3 log10 CFU/ml or 0.1 ng/ml of DNA. However, upon enrichment, the PCR was able to detect 2 log10 and 1 log10 CFU/ml of A. baumanni in water after 6 h and 14 h of incubation at 37oC, respectively. The RT-PCR developed in this study enabled specific detection of A. baumannii, and may be useful in the rapid detection of the pathogen from environmental and clinical samples.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Deepti Karumathil, Meera Surendran Nair and Kumar Venkitanarayanan. Efficacy of TransCinnamaldehyde and Eugenol in Reducing A. Baumannii Adhesion to and Invasion of Human Keratinocytes and Controlling Wound Infections In Vitro. 2016. American Society for Microbiology annual meeting held in Boston, MA. June 16-20, 2016
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Deepti Prasad Karumathil, James Gaffney, Meera Surendran Nair, Anup Kollanoor-Johny, Kumar Venkitanarayanan. 2016. Trans-Cinnamaldehyde and Eugenol Increase Acinetobacter baumannii Sensitivity to Beta-Lactam Antibiotics. Frontiers in Microbiology (under second review after minor revision).
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Microbiologists and regulatory agencies Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project constituted part of a Ph.D. dissertation of a graduate student, and provided opportunities for the student to present the research findings at a national conference. How have the results been disseminated to communities of interest?This research project was presented in a poster at the 2016 American Society for Microbiology annual meeting held in Boston, MA. June 16-20, 2016. Deepti Karumathil, Meera Surendran Nair and Kumar Venkitanarayanan. Efficacy of Trans-Cinnamaldehyde and Eugenol in Reducing A. Baumannii Adhesion to and Invasion of Human Keratinocytes and Controlling Wound Infections In Vitro What do you plan to do during the next reporting period to accomplish the goals?Optimize a real-time quantitative PCR for rapid and specific detection of A. baumannii in blood and water.
Impacts
What was accomplished under these goals?
Acinetobacter baumannii is a multi-drug resistant, nosocomial pathogen causing a variety of disease conditions in humans. After A. baumannii outbreaks in military combat personnel in Iraq and Afghanistan, reports of A. baumannii wound infections are increasingly recognized. In addition, A. baumannii's ability to form biofilms and colonize epithelial cells potentially increases the invasiveness of this pathogen. This study investigated the efficacy of two natural, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde (TC) and eugenol (EG) for decreasing A. baumannii adhesion to and invasion of normal human keratinocytes (HEK001). The efficacy of two PDAs for inhibiting A. baumannii biofilm formation was also determined using an in vitro collagen matrix wound model. In addition, the effect of TC and EG on A. baumannii biofilm architecture was visualized using confocal scanning microscopy. Furthermore, the effect of both PDAs on A. baumannii genes critical for biofilm synthesis was determined using real-time quantitative PCR (RT-qPCR). Both TC and EG significantly reduced A. baumannii adhesion to HEK001 by ~2 to 2.5 log10 CFU/ml (P < 0.05), and invasion by ~2 to 3 log CFU/ml, compared to the controls (P < 0.05). Further, after 24 and 48 h, TC inhibited biofilm formation by ~1.5 and ~2 to 3.5 log10 CFU/ml, while EG decreased biofilm-associated bacteria by ~2 and ~3.5 log10 CFU/ml, respectively, compared to controls (P < 0.05). Confocal microscopy revealed that TC and EG resulted in the death of biofilm-associated A. baumannii, and disrupted the biofilm architecture. RT-qPCR results indicated that the two phytochemicals significantly down-regulated the transcription of genes associated with A. baumannii biofilm production.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Deepti P. Karumathil,Meera Surendran-Nair, and Kumar Venkitanarayanan. 2016. Efficacy of Trans-cinnamaldehyde and Eugenol in Reducing Acinetobacter baumannii Adhesion to and Invasion of Human Keratinocytes and Controlling Wound Infection In Vitro. Phytotherapy Research, 30 (12):20532059
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Microbiologists, Regulatory Agencies Changes/Problems:Instead of the original objective 2 (Develop and optimize PCR for rapid detection of A.baumannii), objective 3 investigating the efficacy of sub-inhibitory concentrations of food-grade, plant-derived animicrobials (PDAs) on decreasing antibiotic resistance in A. baumannii was accomplished during the past year. The objective on developing a PCR for detecting A. baumannii has already begun and will be completed this year. What opportunities for training and professional development has the project provided?The project constituted a part of the Ph.D. dissertation of a graduate student, and provided opportunities for training the student in detecting antibiotic and chlorine resistance in A. baumannii using both traditional and molecular techniques. The project also provided opportunities for the student to present the research findings at a national conference. How have the results been disseminated to communities of interest?One poster each was presented at the Annual Meeting of the Institute Food Technologists held in June 2014 at New Orleans, and Annual meeting of the American Society for Miocrobiology held in May 2014 at Boston. What do you plan to do during the next reporting period to accomplish the goals?Develop and optimize PCR for rapid detection of A.baumannii. Publish the results in a peer-reviewed journal.
Impacts
What was accomplished under these goals?
Effect of Trans-cinnamaldehyde and Eugenol on decreasing A. baumannii resistance to β-lactam antibiotics. This study investigated the efficacy of two food-grade, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde(TC) and eugenol (EG) in decreasing A.baumannii's resistance to seven β-lactam antibiotics, including ampicillin, methicillin, meropenem, penicillin, aztreonam, amoxicillin and piperacillin. Two MDR A.baumannii isolates (ATCC 17978 and AB251847) were separately cultured in tryptic soy broth (~6logCFU/ml) containing the minimum inhibitory concentration (MIC) of TC or EG with or without the MIC of each antibiotic at 37oC for 18 h. A.baumannii strains not exposed to the PDAs or antibiotics served as controls. Following incubation, A.baumannii counts were determined by broth dilution assay. In addition, the effect of PDAs on the permeability of outer membrane and efflux pumps in A. baumannii was measured. Further, the effect of TC and EG on the expression of A.baumannii genes encoding resistance to β-lactam antibiotics (blaP), efflux pumps (adeABC) and multi-drug resistant protein (mdrp) was studied using real-time quantitative PCR (RT-qPCR). The experiment was replicated three times with duplicate samples of each treatment and control. The results from broth dilution assay indicated that both TC and EG in combination with antibiotics increased the sensitivity of A.baumannii to all the tested antibiotics (P<0.05). The two PDAs inhibited the function of A. baumannii efflux pump, (AdeABC), but did not increase the permeability of its outer membrane. Moreover, RT-qPCR data revealed that TC and EG down-regulated the expression of majority of the genes associated with β-lactam antibiotic resistance, especially blaPand adeABC (P<0.05).The results suggest that TC and EG could potentially be used along with β-lactam antibiotics for controlling MDR A.baumannii infections; however, their clinical significance needs to be determined using in vivo studies.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
D. P. Karumathil, H. Yin, A. Kollanoor-Johny and K. Venkitanarayanan. Prevalence of multi-drug resistant bacteria in vegetables collected from farmers markets in Connecticut. Presented at the 2014 IFT Annual meeting held in June 2014 at New Orleans, LA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
D. P. Karumathil, Anup Kollanoor-Johny, and Kumar Venkitanarayanan. Effect of Trans-cinnamaldehyde and Eugenol in reducing the resistance of MDR Acinetobacter baumannii to Beta-lactam Antibiotics. Presented at the 114th ASM General meeting held in May 2014 at Boston, MA.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Microbiologists, Regulatory Agencies Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project constitutes a part of the Ph.D. dissertation of a graduate student, and provided opportunities for training the student in detecting antibiotic and chlorine resistance in A. baumannii using both traditional and molecular techniques. The project also provided opportunities for the student to present the research findings at a national conference. How have the results been disseminated to communities of interest? The results have been disseminated to the scientific community through a peer-reviewed journal manuscript and a conference presentation. Karumathil, D.P.; Yin, H.-B.; Kollanoor-Johny, A.; Venkitanarayanan, K. Effect of chlorine exposure on the survival and antibiotic gene expression of multidrug resistantAcinetobacter baumanniiin water.Int. J. Environ. Res. Public Health. 2014,11, 1844-1854. Karumathil, D.P.; Yin, H,; Kollanoor-Johny, A.; Venkitanarayanan, K. (2013). Effect of chlorine on the viability of the multidrug resistant Acinetobacter baumannii in water and expression of antibiotic resistance genes. Presented at the 113th ASM General meeting, held in May 2013 at Denver, CO. What do you plan to do during the next reporting period to accomplish the goals? To study the effect of plant derived antimicrobials in reducing antibiotic resistance of A. baumannii. To develop a PCR to detect A. baumannii.
Impacts
What was accomplished under these goals?
Investigated the effect of chlorine on the survival of multi-drug resistant Acinetobacter baumannii, and expression of its antibiotic resistance genes. This study investigated the effect of different levels of chlorine on the viability of A. baumannii in water. Additionally, the effect of chlorine exposure on the transcription of genes conferring antibiotic resistance in A. baumannii was studied. Eight clinical isolates of A. baumannii were separately exposed to different chlorine concentrations (0.2, 1, 2, 3, and 4 ppm) with a contact time of 30, 60, 90 and 120 sec. The surviving pathogen counts at each specified contact time were determined using broth dilution assay. In addition, the expression of A. baumannii efflux pump genes (adeA, adeB, adeC, abeM), and those encoding resistance to antibiotics, including β-lactams (blaP), aminoglycoside (ami), chloramphenicol (cmr), sulfamethoxazole (sul1), tetracycline (tetA), and multiple drug resistance protein (mdrp) was determined by real-time quantitative PCR (RT-qPCR) in three isolates following exposure to chlorine. Major Findings: The eight A. baumannii isolates survived all the tested chlorine levels during the entire duration of exposure period. Moreover, RT-qPCR results revealed that the expression of all the tested antibiotic resistance genes, except tetA was up-regulated in two strains of A. baumannii, whereas adeC, cmr, tetA were up-regulated in the third strain (P < 0.05). The results revealed that chlorine at the tested concentrations is not only ineffective in significantly reducing A. baumannii in water, but also induces the up-regulation of genes conferring resistance to multiple antibiotics in the pathogen.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Karumathil, D.P.; Yin, H.-B.; Kollanoor-Johny, A.; Venkitanarayanan, K. Effect of chlorine exposure on the survival and antibiotic gene expression of multidrug resistant Acinetobacter baumannii in water. Int. J. Environ. Res. Public Health. 2014, 11, 1844-1854.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Food Safety Scientists, Food Industry, Regulatory Agencies, Public Health Scientists Changes/Problems: None What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? An abstract of the research has been submitted for presentation at the Annual Meeting of the INstitute Food Technologists to be held in June 2014 at New Orleans. What do you plan to do during the next reporting period to accomplish the goals? 1. Develop and optimize PCR for rapid detection of A.baumannii. 2. Publish the results from objective 1 in a peer-reviewed journal.
Impacts
What was accomplished under these goals?
A total of 300 samples of lettuce, carrots and potatoes (n=100) were collected from 14 farmers markets in Connecticut, and tested for the presence of antibiotic resistant bacteria, including A. baumanniiby plating. Among the produce tested, 73% lettuce, 86% carrots and 74% potatoes were found to harbor bacteria resistant to multiple antibiotics. These bacteria were isolated in pure cultures and identified using API 20NE as Burkholderia cepacia/gladioli (48%), Pseudomonas luteola (41%), Stenotrophomonas maltophilia (18%) and Acinetobacter baumannii/calcoaceticus (1.3%). In addition, the antibiotic resistance profile of each of these isolates was determined using antibiotic disc diffusion assay. High resistance rates were observed for ceftriaxone (90%), streptomycin (85%) and neomycin (72%) in Burkholderia cepacia/gladioli; for imipenem (10%) and colistin-sulphate (30%) in Pseudomonas luteola; for doxycycline (56%), imipenem (70%), erythromycin (80%) and minocycline (41%) in Stenotrophomonas maltophilia, and for imipenem (75%), rifampcin (25%), streptomycin and ceftriaxone (100%) in Acinetobacter baumannii/calcoaceticus. The results reveal the high prevalence of multi-drug resistant bacteria, including A. baumannii on fresh produce, thereby highlighting the potential health impact in consumers, especially those with a compromised immune system.
Publications
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