Understanding and Harnessing Molecular Barriers to Vancomycin Resistance in Agriculture

<p>NON-TECHNICAL SUMMARY: <br/>The global emergence of antimicrobial resistant bacteria has serious consequences for both human and animal health. Food animal agriculture has been blamed for decades by both national and international advocacy groups as the major source of antibiotic resistance among food borne enteric bacterial pathogens. More often, these enteric pathogens harbor resistances to multiple antibiotics which have a greater propensity for horizontal gene transfer. These resistant bacteria continue to propagate and their increased prevalence poses significant clinical and societal challenges. Our proposal focuses on one of the major clinically important enteric bacterial pathogens, Enterococcus. The enterococci are intrinsically resistant to broad range of antimicrobials agents, thus leading to a pre-existing limited choice of antimicrobials against these agents.
During the last few decades, enterococci have emerged as one of the important pathogens attributed to significant causes of community acquired and nosocomial infections. The role of foodborne transfer of enterococci remains controversial; however, recent discoveries of VRE among swine in Michigan have refocused concern on this potential mode of transfer. Our hypothesis is novel and concentrates on the impact of feeding heavy metals and their possible role in dissemination of vancomycin resistant enterococci (VRE) and also their selection pressure against heavy metal resistance. We have reported the presence of transferable copper resistance gene, tcrB among enterococcal isolates of swine with its co-selection of macrolide and tetracycline resistant determinants, for the first time in the US. Therefore, the bacteria that are resistant to heavy metals, more often than not also carry other
antibiotic resistant determinants. The major objectives of our proposal includes characterization of subset of enterococcal isolates belonged to Texas and Kansas. The Texas study consisted of isolates arising from groups of swine and human in semi-closed integrated agri food system. The Kansas study consists of both copper resistant and sensitive isolates of swine. To accomplish our objectives we will use traditional bacteriological procedures coupled with cutting edge molecular biology techniques.
<p>APPROACH: <br/>The approach to accomplish these objectives involves utilization of enterococcal isolates from studies conducted at two geographical locations in Kansas and Texas. The Texas study resulted in isolates arising from groups of swine and human in a semi-closed and fully integrated agri-food system. The population of both swine and humans were housed across the multiple locations. Humans were further categorized into swine-worker and non-worker groups. VRE were only isolated from humans (both swine-worker and non-workers). Non-vancomycin-resistant enterococci (vancomycin-susceptible enterococci - VSE) were isolated from swine fecal samples, floor wash, influent, lagoon samples, and human waste water grab samples. The Kansas study consisted of both copper resistant and sensitive isolates of swine from a series of feeding trials. To accomplish our objectives we
will use traditional bacteriological procedures coupled with cutting edge molecular biology techniques. Earlier studies from our laboratory reported the presence of tcrB (transmissible copper resistance) gene from fecal enterococcal isolates of swine for the first time in US. There are strong associations between tcrB and erm(B) (macrolide) and tet(M) (tetracycline). Further research is warranted to investigate the epidemiology of VRE in U.S and its dissemination into food producing animals to elucidate the public health significance of VRE within the integrated agri-food system. The proposed work will be carried out to study the dynamics of transfer and its frequency among enterococci with in closed population of human and animals. The study will be mainly focused on determining transferability of antibiotic resistant determinants from human enterococci to animal enterococcal isolates.
The isolates will be subjected to antibiotic resistance determinants assay and characterization by PCR, RT-PCR for quatification, susceptibility determination (MIC), conjugation assay to determine the transferability of resistant determinants, and multi-locus sequence typing to determine the clonal differences among the clinically predominant isolates. This will help us to elucidate the global epidemiological distribution of predominant sequence types and clonal complexes prevalent among VRE's in closed population of humans and animals and their zoonotic potential. The molecular epidemiological studies and population structures analysis will give us an idea whether the emergence of E. faecium and E. faecalis as major nosocomial pathogens has resulted from the evolutionary development of specific lineages or clonal complexes harboring antimicrobial resistance determinants. The statistical
analyses will be performed using STAT SE (v. 11.0). Descriptive, bivariate, and multivariate analyses will be used in a multi-level mixed model framework for each of the outcomes, such as growth curve, plasmid transfer efficiency etc.
<p>PROGRESS: 2013/01 TO 2013/09<br/>Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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
<p>PROGRESS: 2012/01/01 TO 2012/12/31<br/>OUTPUTS: The objectives of this project were to investigate the prevalence of vancomycin resistant enterococci (VRE; vanA), determine the genetic relationship between van genes and other antimicrobial resistance determinants [erm(B) and tet(M)] among both human and swine enterococcal isolates. The study design consisted of isolates arising from groups of humans and swine in a semi-closed, vertically integrated agri-food system. The isolates were from swine fecal samples, floor wash, influent, lagoon samples, and human waste water grab samples. We have approximately 2,500 isolates (1,252 human + 1,270 swine isolates), of which 62 isolates were positive for vanA and resistant to vancomycin among human isolates. One isolate was positive for both vanA and vanB genes. All these 62 isolates were also positive for the presence of erm(B)
and tet(M) genes and also phenotypically resistant to erythromycin and tetracycline. Most of the isolates (57) harbored esp gene and one isolate was positive for both esp and hyl, virulence genes. The conjugation experiment proved that VREs are transferable and resides on a plasmid, mobile genetic element. All these isolates were Enterococcus faecium. Among 486 swine enterococcal isolates screened till now, 6 were positive for vanA gene and resistant to vancomycin. All these VRE isolates were also harbored erm(B) and tet(M) genes. The presence of vanA gene was further confirmed by sequence analysis. The study was further extended to study the sequence types and clonal complexes by multi-locus variable number tandem repeat (MLVA) analyses for both human and swine VRE isolates to assess their significance in relation to global prevalence of VRE, and also to determine the presence of
virulence genes and to explore the possibility of linkage of these virulence factors with antibiotic resistance determinants and clonal differences among VREs. MLVA profile was submitted to the database maintained at the University of Utrecht, The Netherlands, to get MTs or STs numbers. MLVA analysis revealed the presence of diverse and heterogeneous clones of enterococcal isolates. At present, we are in the process of performing descriptive statistics on the prevalence data, generating eBURST analysis and creating a phylogenetic tree based on the assigned STs or MTs. PARTICIPANTS: Dr. H. Morgan Scott (Kansas State University), Principal Investigator Collaborators: Dr. T. G. Nagaraja (Kansas State University) Dr. R. B. Harvey (Southern Plains Agricultural Research Center, ARS, USDA, College Station, Texas) Dr. T. L. Poole (Southern Plains Agricultural Research Center, ARS, USDA, College
Station, Texas) Graduate Student: Raghavendra Amachawadi Undergraduate Students: Tiffany Mainini, Melody Phillips TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2011/01/01 TO 2011/12/31<br/>OUTPUTS: The objectives of this project were to investigate the prevalence of vancomycin resistant enterococci (VRE; vanA), determine the genetic relationship between van genes and other antimicrobial resistance determinants [erm(B) and tet(M)] among both human and swine enterococcal isolates. The study design consisted of isolates arising from groups of humans and swine in a semi-closed, vertically integrated agri-food system. The isolates were from swine fecal samples, floor wash, influent, lagoon samples, and human waste water grab samples. We have approximately 2,500 isolates (1,252 human + 1,270 swine isolates), of which 62 isolates were positive for vanA and resistant to vancomycin among human isolates. One isolate was positive for both vanA and vanB genes. All these 62 isolates were also positive for the presence of erm(B)
and tet(M) genes and also phenotypically resistant to erythromycin and tetracycline. Most of the isolates (57) harbored esp gene and one isolate was positive for both esp and hyl, virulence genes. The conjugation experiment proved that VREs are transferable and resides on a plasmid, mobile genetic element. All these isolates were Enterococcus faecium. Among 370 swine enterococcal isolates screened till now, 6 were positive for vanA gene and resistant to vancomycin. All these VRE isolates were also harbored erm(B) and tet(M) genes. At present, we are still in the process of screening remaining swine fecal samples for the presence of VREs. The study will be further extended to study the sequence types and clonal complexes by multi-locus sequence typing (MLST) analyses to assess their significance in relation to global prevalence of VRE, and also to determine the presence of virulence genes
and to explore the possibility of linkage of these virulence factors with antibiotic resistance determinants and clonal differences among VREs. Our future studies might give us an idea on evolutionary development of specific lineages or clonal complexes harboring multidrug resistance determinants. PARTICIPANTS: Principal Investigator: Dr. H. Morgan Scott (Kansas State University) Collaborators: Dr. T. G. Nagaraja (Kansas State University) Dr. R. B. Harvey (Southern Plains Agricultural Research Center, ARS, USDA, College Station, Texas) Dr. T. L. Poole (Southern Plains Agricultural Research Center, ARS, USDA, College Station, Texas) Graduate Student: Raghavendra Amachawadi Undergraduate Students: Tiffany Mainini, Melody Phillips TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2011/01/01 TO 2011/12/31<br/>OUTPUTS: The objectives of this project were to investigate the prevalence of vancomycin resistant enterococci (VRE; vanA), determine the genetic relationship between van genes and other antimicrobial resistance determinants [erm(B) and tet(M)] among both human and swine enterococcal isolates. The study design consisted of isolates arising from groups of humans and swine in a semi-closed, vertically integrated agri-food system. The isolates were from swine fecal samples, floor wash, influent, lagoon samples, and human waste water grab samples. We have approximately 2,500 isolates (1,252 human + 1,270 swine isolates), of which 62 isolates were positive for vanA and resistant to vancomycin among human isolates. One isolate was positive for both vanA and vanB genes. All these 62 isolates were also positive for the presence of erm(B)
and tet(M) genes and also phenotypically resistant to erythromycin and tetracycline. Most of the isolates (57) harbored esp gene and one isolate was positive for both esp and hyl, virulence genes. The conjugation experiment proved that VREs are transferable and resides on a plasmid, mobile genetic element. All these isolates were Enterococcus faecium. Among 370 swine enterococcal isolates screened till now, 6 were positive for vanA gene and resistant to vancomycin. All these VRE isolates were also harbored erm(B) and tet(M) genes. At present, we are still in the process of screening remaining swine fecal samples for the presence of VREs. The study will be further extended to study the sequence types and clonal complexes by multi-locus sequence typing (MLST) analyses to assess their significance in relation to global prevalence of VRE, and also to determine the presence of virulence genes
and to explore the possibility of linkage of these virulence factors with antibiotic resistance determinants and clonal differences among VREs. Our future studies might give us an idea on evolutionary development of specific lineages or clonal complexes harboring multidrug resistance determinants. PARTICIPANTS: Principal Investigator: Dr. H. Morgan Scott (Kansas State University) Collaborators: Dr. T. G. Nagaraja (Kansas State University) Dr. R. B. Harvey (Southern Plains Agricultural Research Center, ARS, USDA, College Station, Texas) Dr. T. L. Poole (Southern Plains Agricultural Research Center, ARS, USDA, College Station, Texas) Graduate Student: Raghavendra Amachawadi Undergraduate Students: Tiffany Mainini, Melody Phillips TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.