Oxytetracycline Resistant Gram-Negative Bacteria in Dairy Cattle: Risk Factors and Implications on Food Safety

OXYTETRACYCLINE RESISTANT GRAM-NEGATIVE BACTERIA IN DAIRY CATTLE: RISK FACTORS AND IMPLICATIONS ON FOOD SAFETY

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

TERMINATED

Funding Source

NRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

0192991

Grant No.

2002-35201-12474

Project No.

PEN03904

Proposal No.

2002-02589

Multistate No.

(N/A)

Program Code

32.0

Project Start Date

Sep 1, 2002

Project End Date

Aug 31, 2005

Grant Year

2002

Project Director
Jayarao, B. M.

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802

Performing Department
VETERINARY SCIENCE

Non Technical Summary
The development of resistance to tetracyclines in the context of human and animal use has raised great concern. Tetracyclines have been used in humans and animals for more than four decades. This has resulted in the emergence of resistant bacterial variants, particularly those containing tetracycline resistant genes, observed most frequently in multi-drug resistant gram-negative bacteria. In recent years the use of oxytetracyclines as animal growth promoters and the implications for human health has been questioned. The purpose of this study is to gather relevant and accurate information on the epidemiology (descriptive and molecular) of oxytetracycline resistant gram-negative bacteria in dairy cattle. This study will provide much needed scientific information with reference to prevalence, trends of antimicrobial resistance on the farm with respect to oxytetracycline and other antimicrobial agents, identify risk factors for resistance development, and develop and implement interventions to reduce risk for resistance. A clear understanding of 'critical' farm management practices will allow us in the long term to: (1) develop and implement hazard analysis critical control point-based programs to address issues related to antimicrobial-resistant bacteria, and food borne pathogens on dairy herds, and (2) provide a strong and pertinent knowledge base to undertake risk analysis investigations.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3410	1100	50%
712	3410	1170	50%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1170 - Epidemiology; 1100 - Bacteriology;

Goals / Objectives
Objective 1: Conduct a prospective longitudinal study to assess the effect of feeding milk replacers (with and without oxytetracyline) to calves on: (1) fecal shedding of oxytetracycline resistant gram-negative bacteriaOXY-GNB in calves and lactating cattle, (2) farm environmental contamination with OXY-GNB, and (3) contamination of bulk tank milk (BTM) with OXY-GNB. Objective 2: Conduct a survey to determine the prevalence and distribution of OXY-GNB, shiga-toxin producing E. coli, and salmonellae in feces of lactating cattle and BTM. Objective 3: Identify farm management practices related 'risk factors' that influence OXY-GNB fecal shedding in lactating cows and BTM contamination.

Project Methods
The first objective of the study is to determine the outcome of feeding or withdrawing milk replacers to calves supplemented with subtherapeutic concentrations of oxytetracycline to calves. A prospective longitudinal study will be conducted on 8 dairy farms. Feces from calves and lactating cattle will be collected. Environmental swabs and bulk tank milk will also be collected. Samples will be collected periodically from each farm for duration of 300 days. The samples will be examined for oxytetracycline gram-negative bacteria. (OXY-GNB). Samples will be plated on MacConkey's agar supplemented with oxytetracycline. Bacteria that grow on the plates will be speciated using API-20E identification system. OXY-GNB bacteria will be subjected to antimicrobial susceptibility testing using the methods described for dilution antimicrobial susceptibility tests for aerobic bacteria by National Committee for Clinical Laboratory Standards. The OXY-GNB bacteria will be subtyped using the pulsed field gel electrophoresis (PFGE) technique. The PFGE protocol followed in our laboratory is based on Centers for Disease Control and Prevention recommended procedures. The genetic determinants of tetracycline resistance will be done using standardized protocols developed for tetracycline resistance analyses. In our laboratory, we have successfully analyzed tetracycline determinants in Escherichia coli and Salmonella isolates. The second objective of the study is to determine the prevalence and distribution of OXY-GNB, shiga toxin producing Escherichia coli and Salmonella in feces of lactating cattle and bulk tank milk. A survey will be conducted using the guidelines established by the National Animal Health Monitoring System'98 study. Under this objective, dairy herds located in 67 counties in Pennsylvania will be surveyed. The questionnaire will be administered to the dairy producer during the farm visit. The survey instrument has been designed to elicit both quantitative and or qualitative responses, which can be scored and used to assess farm management practices including use of antimicrobial agents. A total of 160 farms will be surveyed. Bulk tank milk and fecal samples from approximately 10% of the animals on each herd will be collected and examined for OXY-GNB, shiga-toxin producing E. coli and Salmonella. Isolation, identification and characterization of OXY-GNB. Shiga-toxin producing E. coli and Salmonella will be done as described under objective 1. Isolates identified as belonging to the genus. Escherichia will be tested for shiga-toxins stx(1) and stx(2) by a specific PCR assay. The third objective of the study is to utilize the data collected from objective 1 and 2 to determine the risk factors for OXY-GNB, shiga toxin producing E. coli and Salmonella in dairy herds. Data will be entered and maintained on Microsoft Excel. Appropriate statistical analysis will be done to answer the research questions listed under objective 1 and 2. A scheme termed as interactive Dichotomizer 3 will be used to induce decision trees for classification tasks to develop the neural network model. The results of this study will be the development of a prototype software.

Progress 01/01/05 to 12/31/05

Outputs
Under the first objective, a prospective longitudinal study was conducted to assess the effect of withdrawing the use of oxtyetracyline. Withdrawal of oxytetracycline as prophylactic and therapeutic agent for 12 months did not result in a significant decrease in OXY-GNB counts in the feces of calves and lactating cows, environmental samples and bulk tank milk. Modeling for effect of temperature and rainfall on the number of OXY-GNB in calves and cattle, clearly showed that an increase in OXY-GNB counts was associated with an increase in the ambient temperature. Under the second objective, 67 farms from 40 counties in Pennsylvania were surveyed to determine the prevalence and distribution of oxytetracycline resistant gram-negative bacteria (OXY-GNB), shiga-toxin producing E. coli, and salmonellae in feces of lactating cattle and BTM. OXY-GNB E. coli was isolated from 49 of 67(73%) farms and 429 of 693 (62%) lactating cattle. While shiga toxin producing OXY-GNB E. coli was isolated from 5% and 12% of the farms and lactating cows respectively. The OXY-GNB E. coli including shiga toxin producing E. coli belonged to 15 different serotypes, and 93 PFGE types, suggestive of the fact that OXY-GNB E. coli are extremely heterogeneous. OXY-GNB shiga-toxin producing E. coli was isolated from 2% of the bulk tank milk samples. The genotypes of these organisms were similar to that isolated from feces of lactating cattle. Salmonella was isolated from 11, 8 and 3% of the farms, lactating cattle and bulk tank milk. Salmonella serotypes isolated were. S. Typhimurium, S. Typhimurium var Copenhagen, S. Montevideo, S. Kentucky, S. Muenchen, and S. Newport. Tet determinants tet(B) and tet(A) were detected in 93 and 7% of the isolates, respectively. DNA-DNA hybridization assays revealed that tet determinants were located on the chromosome. A sub-genomic library of E. coli T8 isolate that tetracycline determinant was located on the Tn10 transposon. This is the first report of a chromosomally located tet efflux pump associated with transposon Tn10 in enteric E. coli isolated from lactating cattle. The sequence (4632 bp) can be accessed through NCBI GeneBank (accession no. AY528506). The findings of this study suggest that commensal enteric E. coli from lactating cattle can be a significant reservoir for oxytetracycline-resistance determinants. Under the third objective, risk factors that influence OXY-GNB fecal shedding in lactating cows and contamination of bulk tank milk were determined. Feeding calves with medicated milk replacers containing oxytetracycline and neomycin was the main risk factor. Other factors identified were, 1) widespread prevalence of transferable tetracycline determinants in enteric bacteria,1) transfer of oxytetracyline alone resistance is horizontally mediated at very low frequency, however oxytetracycline resistance is transferred at a considerably higher frequency when ampicillin and related beta-lactams are also encoded by the organism.

Impacts
Results of the study suggest that withdrawal of oxytetracycline as prophylactic (feeding calves) and therapeutic agent (treating cows with infectious disease) for 12 months did not result in a significant decrease the number of oxytetracycline resistant gram-negative bacteria in the feces of calves and lactating cows, environmental samples and bulk tank milk. It was observed that increase in number of oxytetracycline resistant gram-negative bacteria was associated with an increase in the ambient temperature. The results of the study suggest that oxytetracycline resistant gram-negative bacteria, E. coli in particular are widely distributed on dairy herds. Lactating cattle serve as incubators for oxytetrcycline resistant E. coli and allow dissemination of antibiotic resistant bacteria. Only a small percentage (2%) of the shiga toxin producing E. coli and Salmonella were present in bulk tank milk. The findings of the study suggest that tetracycline resistance tet(B) determinant is a part of tn10 gene that is chromosomally located and may be horizontally transferred from one E. coli to other and perhaps other gram negative enteric bacteria. It is inferred that reduction in the number and type of tetracycline resistant bacteria on dairy herds may be difficult to accomplish with the current farm management practices.

Publications

No publications reported this period

Progress 01/01/04 to 12/31/04

Outputs
The second objective of the study has been completed. Under the second objective, 67 farms from 40 counties in Pennsylvania were surveyed to determine the prevalence and distribution of oxytetracycline resistant gram-negative bacteria (OXY-GNB), shiga-toxin producing E. coli, and salmonellae in feces of lactating cattle and BTM. OXY-GNB E. coli was isolated from 49 of 67(73%) farms and 429 of 693 (62%) lactating cattle. While shiga toxin producing OXY-GNB E. coli was isolated from 5% and 12% of the farms and lactating cows respectively. The OXY-GNB E. coli including shiga toxin producing E. coli belonged to 15 different serotypes, and 93 PFGE types, suggestive of the fact that OXY-GNB E. coli are extremely heterogeneous. Interestingly, nearly 81% of the isolates encoded for tet(B) determinant OXY-GNB shiga-toxin producing E. coli was isolated from 2% of the bulk tank milk samples. The genotypes of these organisms were similar to that isolated from feces of lactating cattle. Salmonella was isolated from 11, 8 and 3% of the farms, lactating cattle and bulk tank milk. Salmonella isolates are currently being serotyped. The third objective of the study involves identifying farm management practices related to risk factors that influence OXY-GNB fecal shedding in lactating cows and contamination of bulk tank milk. All of the 67 farms (objective 2) were also surveyed for their farm management practices relating to farm hygiene and management and use of antibiotics. This data set is currently being analyzed to determine risk factors associated with farm hygiene practices and antibiotic usage on dairy herds.

Impacts
Preliminary results of the study suggest that withdrawal of oxytetracycline as a prophylactic (feeding calves) and therapeutic agent (treating cows with infectious disease) for 12 months did not result in a significant decrease the number of oxytetracycline resistant gram-negative bacteria in the feces of calves and lactating cows, environmental samples and bulk tank milk. It was observed that an increase in number of oxytetracycline resistant gram-negative bacteria was associated with an increase in the ambient temperature.

Publications

No publications reported this period

Progress 01/01/03 to 12/31/03

Outputs
The first objective of the study has been completed and the results of the study are currently being analyzed. Under the first objective, a prospective longitudinal study was conducted to assess the effect of withdrawing the use of oxytetracyline as a prophylactic (oxytetracycline in milk replacers) and therapeutic agent (treating lactating cattle with oxytetracycline) on the number of oxytetracycline resistant gram negative bacteria in the feces of calves and cattle, environmental samples and bulk tank milk. Use of oxytetracycline was discontinued on 6 dairy herds. All 6 herds were sampled each month for a duration of 12 months for OXY-GNB. Preliminary results of the study suggest that withdrawal of oxytetracycline as a prophylactic and therapeutic agent for 12 months did not result in a significant decrease in OXY-GNB counts in the feces of calves and lactating cows, environmental samples and bulk tank milk. Interestingly modeling for effect of temperature and rainfall on the number of OXY-GNB in calves and cattle, clearly showed that an increase in OXY-GNB counts was associated with an increase in the ambient temperature. Based on these preliminary results it can be inferred that withdrawal of oxytetracycline did not result in significant decrease in the number of OXY-GNB. There could be several other factors such as weather conditions that influence OXY-GNB counts in the feces of calves and cattle and the farm environment. The second objective of the study will begin in the month of April 2004. In this study, 67 farms from 40 counties in Pennsylvania will be surveyed to determine the prevalence and distribution of OXY-GNB, shiga-toxin producing E. coli, and salmonellae in feces of lactating cattle and BTM.

Impacts
Preliminary results of the study suggest that withdrawal of oxytetracycline as a prophylactic (feeding calves) and therapeutic agent (treating cows with infectious disease) for 12 months did not result in a significant decrease the number of oxytetracycline resistant gram-negative bacteria in the feces of calves and lactating cows, environmental samples and bulk tank milk. It was observed that an increase in number of oxytetracycline resistant gram-negative bacteria was associated with an increase in the ambient temperature.

Publications

No publications reported this period

Progress 01/01/02 to 12/31/02

Outputs
The findings of the study will provide information on the use of oxytetracycline in milk replaces and its effect on developing antibiotic resistant bacteria in dairy cattle.

Impacts
Work on objective 1 has been initiated. A total of 8 farms are participating in the study. All of the 8 farms have been visited on two separate occasions and samples have been collected and analyzed for oxytetracycline gram-negative bacteria for establishing baseline counts. The farms will be assigned to their respective groups staring March 1, 2003.

Publications

No publications reported this period